CN107278155A - Radiopharmaceutical complex compound - Google Patents

Abstract

The present invention provides a kind of method for the thorium complex for forming tissue targeting, and methods described includes：A) formed and include four at N by C₁‑C₃The octadentate chelating agent of alkyl-substituted Hydroxypyridinone (HOPO) partly with the coupling moiety that end is hydroxy-acid group；B) the octadentate chelating agent is coupled in the peptide or protein matter of at least one tissue targeting comprising at least one amine moiety by least one am amide coupling agent, so as to produce the chelating agent of tissue targeting；And c) chelating agent by the tissue targeting is contacted with the aqueous solution of the thorium isotope ion comprising at least one transmitting α.A kind of method for treating tumprigenicity or proliferative disease is also provided, it includes the thorium complex for giving this kind of tissue targeting；And the complex compound and corresponding pharmaceutical preparation.

Description

Radiopharmaceutical complex compound

Technical field

The present invention relates to the complex compound for forming thorium isotope --- and particularly thorium -227 is conjugated to what tissue was targetted with some The complex compound of partial octadentate part --- method.The invention further relates to the complex compound, and it is related to disease --- particularly Tumor disease --- treatment, it includes giving this kind of complex compound.

Background of invention

In mammalian subject, specific cell killing is probably necessary for the various diseases of successful treatment.Its In treatment of the typical examples for malignant disease such as sarcoma and cancer.It can also treated however, the selectivity of some cell types is eliminated Played a crucial role in other diseases --- being particularly Hypertrophic and tumor disease ---.

At present, the most common method of selective therapy is operation, chemotherapy and external beam irradiation.However, targeting radioactive nucleus Plain therapy is a field for having prospect and developing, and it has specificity high for the cell type delivering relevant with disease The potentiality of cytotoxicity radiation.Authorize at present and use transmitting β and/or hair for the most common form of the radiopharmaceutical in human body Penetrate γ radionuclide.However, due to launching α radionuclide to the potentiality of more specific cell killing, people couple In the treatment some interest are have using transmitting α radioactive nucleus.

The radiation scope for the alpha emitter commonly used in physiological environment is typically smaller than 100 microns, equivalent to only several cells Diameter.This causes these sources to be perfectly suitable for treating tumour, including micrometastasis knurl, because they have the phase for reaching intra-tumor The scope of adjacent cell, but if they have good targeting, then seldom emittance will pass through target cell.Therefore, It need not target each cell, but the damage of surrounding health tissue will can be minimized (referring to Feinendegen et al., Radiat Res 148:195-201(1997)).On the contrary, β particles in water with more than 1mm scope (referring to Wilbur, Antibody Immunocon Radiopharm 4:85-96(1991))。

Compared with the energy that β particles, gamma-rays and X-ray are carried, the energy of alpha-radiation is high, usually 5-8MeV, Or be 5 to 10 times of energy of β particles and be more than 20 times of gamma-ray energy.Therefore, it is big in very short distance This deposition of energy makes alpha radiation be compared with γ and β radiation with extra high linear energy transfer (LET), high relative Biological effect (RBE) and low oxygen enhancement ratio (OER) (referring to Hall, " Radiobiology for the radiologist ", 5th edition, Lippincott Williams＆Wilkins, Philadelphia PA, USA, 2000).This explains transmitting α's The special cytotoxicity of radionuclide, and the biological targeting also to this kind of isotope and the radioactive nucleus to launching α Controlled level and research and propose strict requirements that element is distributed, this is required, to avoid unacceptable side effect.

Table 1 below shows that the possibility proposed extensively in document has the physical decay properties of the alpha emitter of therapeutic efficiency.

Table 1

Candidate nuclides	T1/2*	To following clinical test
			225Ac	10.0 days	Leukaemia
211At	7.2 hour	Spongioblastoma
			213Bi	46 minutes	Leukaemia
223Ra	11.4 days	Skeletal metastasis
			224Ra	3.66 my god	Ankylosing spondylitis

* half-life period

So far, on the application in RIT, main focus is concentrated on²¹¹At、²¹³Bi and²²⁵Ac, And these three nucleic were probed into clinical immunotherapy experiment.

The existence time for several radionuclides having been proposed that is short, i.e. half-life period is less than 12 hours.This short half-life period Make it difficult to that the radiopharmaceutical based on these radionuclides is produced and distributed with commercial system.Give existence time short core Element also increases the ratio for the dose of radiation launched in vivo before target site is reached.

In many cases, from the α recoil energies launched the position from parent decay will be caused to discharge daughter nuclide.Should Recoil energy, which is enough to make many daughter nuclides to depart from, can keep the chemical environment of parent --- for example wherein parent by part such as Chelating agent is complexed.Can also occur this feelings even if daughter (can be by identical ligand complex) compatible with identical ligand chemical Condition.Similarly, when daughter nuclide is gas particularly inert gas such as radon, or when incompatible with ligand chemical, the release Effect will be bigger.When the half-life period of daughter nuclide being more than the several seconds, they can spread in hematological system, not female by holding The limitation of the complexing agent of body.Then, these free radioactive daughters can cause undesirable general toxicity.

Several years ago, propose in holding pair²²³(the T of thorium -227 is used under conditions of the control of Ra daughter isotopes_1/2=18.7 My god) (referring to WO 01/60417 and WO 02/05859).This is that this causes daughter nuclide quilt in the case of using carrier system It is retained in closed environment.In one case, radionuclide is placed in liposome, and the substantial dimensional of liposome (compared with the distance that recoils) helps to retain the daughter nuclide in liposome.In the latter case, using radionuclide Bone affinity (bone-seeking) complex compound, it is bound in bone matrix, so as to limit the release of daughter nuclide.These are latent Very favorable method, but it is not desired to give liposome in some cases, and there are many soft tissue diseases, Wherein radionuclide can not be mineralized matrix and surround to retain daughter isotope.

Recently, it has been determined that, in mammal body, when²²⁷What Th discharged when decaying²²³The toxicity of Ra daughter nuclides can The degree being resistant to is more much bigger than the toxicity tolerance degree predicted by the previous test to comparable core.On reservation is lacked In the case of the specific method of radium daughter for stating thorium -227, the publicly available information about radium toxicity is clearly illustrated, it is impossible to Using thorium -227 as therapeutic agent, because the dosage needed for realizing the therapeutic effect decayed from thorium -227 will cause to come from radium The radiation of the high toxicity of body decay and possible lethal dose, i.e., do not treat window phase.

WO 04/091668 describes unexpected discovery, that is, is implicitly present in treatment window phase, wherein can be to subject (be usually mammal) give the radionuclide of targeting thorium -227 of therapeutically effective amount without produce be enough to cause it is unacceptable Bone marrow toxicity radium -223 amount.Therefore, this all types of disease that can be used for treating and preventing bone and soft tissue position Disease.

In view of above-mentioned development, now can be in interior radio nuclide therapy using the core of thorium -227 for launching α, without by institute Produce²²³Lethal bone marrow toxicity caused by Ra.Nevertheless, treatment window phase still relative narrowness, and in all situations Under be required for giving the transmitting α that subject is not more than absolute demand amount radio isotope.Therefore, if transmitting α thorium- The degree of reliability that 227 cores are complexed and targetted is high, then the useful exploitation to this new treatment window phase will be greatly enhanced.

Because radionuclide is constantly decayed, it is separating and giving to the time handled between subject used in material It is very important.(seldom step, short temperature can be preferably needed if launching α thorium core in the form of quick and convenient preparation Educate the temperature of phase and/or the non-irreversibly influence stereotropic property of target) be complexed, targetted and/or be administered, then it is also phase When valuable.In addition, the method that can (substantially in aqueous) be carried out in the solvent that need not be removed before administration has Solvent is avoided to evaporate or dialyse the important advantage of step.

If the formulation of pharmaceutical products of thorium mark can be developed, it shows the stability significantly improved, also will be considered as With important value.Ensure to adhere to stable target level of product quality, while it is to pass to enable logistics approach to deliver patient dose Important.It is therefore preferable that having the preparation that minimized radiation is decomposed within the time of 1-4 days.

Have shown to be applied to coordination alpha emitter thorium -277 before octadentate chelating agent containing Hydroxypyridinone group, be used for It is subsequent binding to targeting moiety (WO2011098611).Octadentate chelating agent is described, it is connected comprising four by linking group 3 on to the support based on amine, 2 hydroxy pyrimidine ketone groups, it has the independent reactive group for being used for being conjugated to targeted molecular.On The preferred structure for stating invention includes 3,2 hydroxy pyrimidine ketone groups, and regard isothiocyanic acid ester moiety as preferred antibody component Conjugation chemistry material, as shown in compound ALG-DD-NCS.Isothiocyanates, which is widely used for mark by amido, to be connected To protein.Isothiocyanates group and the amino terminal and primary amine reaction in protein, and have been used for marking many eggs White matter (including antibody).Although the thiocarbamide key formed in these conjugates is considerably stablized, report by the different sulphur of fluorescence Antibody conjugates prepared by cyanate are decayed [Banks PR, Paquette DM., Bioconjug Chem (1995) 6 with the time: 447-458].The thiocarbamide to be formed is reacted by fluorescein isothiocyanate and amine to be also easy to be converted into guanidine in the basic conditions [Dubey I,Pratviel G,Meunier BJournal:Bioconjug Chem(1998)9:627-632].Due to thorium- 227 decay long half time (18.7 days), with reference to the growth half-life period of monoclonal antibody, it is desirable to use more stable connecting portion Point, to produce the conjugate chemically stablized in vivo and more in terms of storage.

Disclosed in WO2013/167754 on Hydroxypyridinone part be conjugated it is maximally related work in the early time, it is and public The part with the water-soluble portion comprising hydroxyalkyl degree of functionality is opened.Due to the reactivity of the hydroxyl of this chelating type, make It is impossible for the activation of Acibenzolar, because multiple competitive reactions occur in succession, causes to produce complexity by esterification Product mixtures.Therefore, WO2013/167754 part must be by substituting chemical substance (such as isothiocyanates) and tissue The albumen coupling of targeting, obtains more unstable thiocarbamide conjugate as described above.In addition, WO2013167755 and WO2013167756 discloses hydroxyalkyl/isothiocyanates conjugate, and it is respectively applied to the antibody of CD33 and CD22 targetings.

The present inventor using the storage and administration that make complex compound now it has been determined that keep more stable under mild conditions Coupling part, by the way that specific chelating agent is coupled into the complex compound that suitable targeting moiety formation tissue is targetted, is subsequently added Launch α thorium ion, can quickly produce complex compound.

The content of the invention

Therefore, in a first aspect, the present invention provides a kind of method for the thorium complex for forming tissue targeting, methods described bag Include：

A) formed and include four at N by C₁-C₃Alkyl-substituted Hydroxypyridinone (HOPO) is partly carboxylic acid group with end The octadentate chelating agent of the coupling moiety of group's (or its protected equivalent)；

B) the octadentate chelating agent is coupled to by least one am amide coupling agent at least one comprising at least one amine In the peptide or protein matter of partial tissue targeting, so as to produce tissue targeting chelating agent；And

C) aqueous solution of the chelating agent and the ion of the thorium isotope comprising at least one transmitting α of the tissue targeting is connect Touch.

In this kind of complex compound (and preferably in all aspects of the invention), thorium ion is generally by including hydroxyl pyrrole The octadentate ligand complex of pyridine ketone, the part and then the part that tissue targeting is connected to via amido link.

Generally, this method will be a kind of synthesizes comprising reactive carboxylic-acid functional based on 3, the octadentate chela of 2 hydroxy pyrimidine ketone The method of mixture, the octadentate chelating agent can be passed through in the form of Acibenzolar (such as N-hydroxy-succinamide ester (NHS esters)) It is activated in itself by in-situ activation or by synthesizing and separating Acibenzolar.

The NHS esters of gained can be used in simple Conjugation step preparing the protein form that the chelating agent of wide scope is modified. In addition, highly stable antibody conjugates are easily marked with thorium -227.This can enter at ambient temperature or close under environment temperature OK, generally with high Radiochemical yield and purity.

The method of the present invention is preferably carried out in aqueous, and in one embodiment, it can be no or basic On do not have to carry out in the presence of (be less than 1 volume %) any organic solvent.

It is preferred that targeting moiety include polyclonal antibody and particularly monoclonal antibody and its fragment.Specifically bind piece Section such as Fab, Fab', F (ab')₂It is conventional fragment with single-stranded specific binding antibody.

The complex compound of the tissue targeting of the present invention can be configured to the medicine for being applied to give the mankind or non-human animal subject Thing.

Therefore, in second aspect, the method that the present invention provides production pharmaceutical preparation, it includes forming group specifically described herein The complex compound of targeting is knitted, a kind of pharmaceutical carrier and/or excipient is then added to less.Suitable carrier and excipient include ability Domain is known and records the buffer of any aspect, chelating agent, stabilizer and other suitable components in this article.

On the other hand, the present invention additionally provides the thorium complex of tissue targeting.This kind of complex compound will have to be led to herein Feature described in, preferred feature particularly as described herein.The complex compound can be formed by any means as described herein Or can be formed.Therefore, this kind of method can produce at least one tissue targeting as described in this paper any aspects or embodiment Thorium complex.

In still even further aspect, the present invention provides the pharmaceutical preparation for including any complex compound as described herein.The system Agent can be formed or can formed by any means specifically described herein, and can include at least one buffer, stabilizer and/or tax Shape agent.The selection of buffer and stabilizer can make them contribute to the complex compound that protective tissue is targetted together from RADIATION DECOMPOSITION. In one embodiment, after preparation preparation after a couple of days, the RADIATION DECOMPOSITION of complex compound is minimum in preparation.This is One important advantage, because which solving the potential problems related with medicine supply logistics to product quality, described problem is The implementation of the technology and the key of practical application.

The present invention is had shown that in a large amount of of the targeting for preparing the site (such as the related acceptor of tumour) for biological targets Thorium mark antibody conjugates in practicality.

Embodiment

In the context of the present invention, " tissue targeting " is used herein to mean that the material (particularly when with this During the form of the complex compound of the tissue targeting described in text) it is preferential by self poisoning (and particularly by any conjugated thorium network Compound is positioned) need the tissue site that it has (for example, to deliver radioactive decay) at least one.Therefore, with without The concentration of the complex compound of equal value of targeting moiety is compared, organize targeting group or part be used for after giving to the subject to by At least one required site provides bigger positioning in examination person's body.In the present case, targeting moiety will be preferably chosen with specificity It is bound to the cell surface receptor related to cancer cell or other acceptors related with tumor microenvironment.

Exist many known with the Hypertrophic target relevant with tumor disease.These targets are included near sick cell Extracellular matrix in some acceptors, cell surface protein, transmembrane protein and the proteins/peptides that find.With tumor disease phase The cell surface receptor of pass and the example of antigen include CD22, CD33, FGFR2 (CD332), PSMA, HER2, mesothelin etc.. In one embodiment, the part (such as peptide or protein matter) of targeting is organized to be selected from CD22, CD33, FGFR2 at least one (CD332), the antigen or acceptor of PSMA, HER2 and mesothelin have specificity.

CD22, or differentiation cluster -22, to belong to the SIGLEC lectin families (immune globulin that SIGLEC=sialic acids are combined White type agglutinin) molecule.

CD33 or Siglec-3 is the transmembrane receptor expressed on the cell of myeloid lineage.

FGFR2 is the acceptor of fibroblast growth factor.It is by being present on No. 10 chromosomes in human body The protein of FGFR2 gene codes.

HER2 is the member of human epidermal growth factor acceptor (HER/EGFR/ERBB) family.

PSMA (PSMA) is the enzyme by FOLH1 (folic acid hydrolase 1) gene code in human body.

Mesothelin, also referred to MSLN, are the protein by MSLN gene codes in human body.

In the present case, particularly preferred tissue targeting bonding agent will be selected to be specifically bound to CD22 acceptors.This can example The binding affinity for the cell for such as comparing non-express CD22 by the binding affinity of the cell to expressing CD22 is high more than 50 times (such as high at least 100 times, preferably at least 300 times) reflects.It is believed that CD22 is in the cell with some disease conditions (as shown in this article) in expression and/or be overexpressed, therefore the CD22 specific-binding agents can be used for make complex compound targeted to this Cell of the class by sickness influence.Similarly, near the cell by sickness influence, tissue targeting moiety, which can be bound on cell, to be deposited Cell surface marker (such as CD22 acceptors).CD22 cell surface markers can on sick cell surface ratio in health Expressed in a larger amount on cell surface, or during growth or duplication than rest period during in a greater amount of earth's surfaces of cell surface Reach.In one embodiment, the bonding agent that CD22 specific tissues can be targetted and disease specific cell surface marker Another bonding agent be applied in combination, so as to obtain dual combination complex compound.The bonding agent of CD-22 tissue targeting is typically peptide Or protein, it is as described herein.

Many aspects of the invention specifically described herein are related to the treatment of disease, particularly for selectively targeting lesion group Knit, and be related to the complex compound suitable for this kind of method, conjugate, medicine, preparation, kit etc..In all aspects, lesion Tissue may be present in internal Single locus (such as in the case of local entities's tumour) or may be present in multiple sites (for example Situation that wherein several joints are influenceed by arthritis or in the case of distributing or metastatic Cancerous disease).

Pathological tissues to be targeted can be located at soft tissue site, positioned at calcified tissue site or can be entirely located in soft tissue, It is entirely located in calcified tissue or may include at least one soft tissue site and/or multiple positions at least one calcified tissue site Point.In one embodiment, at least one soft tissue site is targetted.Target site and disease senesis of disease site can be identical , but or can difference (for example wherein metastatic site is by selectively targeted situation).When being related to more than one site, its It may include origin site or can be multiple secondary sites.

Term " soft tissue " used herein represents the tissue without " hard " mineralized dentin matrix.Specifically, institute herein Soft tissue can be any tissue of skeletal tissue.Correspondingly, " soft tissue disease " used herein represents occur Disease in " soft tissue " used herein.The present invention is especially suitable for treating cancer, therefore " soft tissue disease " includes The cancer, sarcoma, myeloma, leukaemia, lymthoma and the mixed type cancer that occur in any " soft " (i.e. non-mineralising) tissue and This other histioid non-cancerous disease.Carcinous " soft tissue disease " includes the entity tumor and metastatic occurred in soft tissue With micrometastasis tumour.In fact, soft tissue disease may include the soft tissue in same patient primary entity tumor and The metastatic tumo(u)r of at least one soft tissue.Or, " soft tissue disease " only can be made up of primary tumor or only by primary Tumour constitutes for the metastatic tumor of skeletal diseases.The present invention all appropriate aspects in, be particularly suitable for treating and/or target be The tumor disease of neoplastic hematologic disorder and particularly lymphoid cell such as lymthoma and lymphoid leukemia, including non-Hodgkin's lymph Knurl, the B cell tumour of B cell lymphoma.Similarly, marrow, backbone (particularly spinal cord) lymph node and/or haemocyte is any Tumor disease is suitable for treatment and/or targeting in all appropriate aspects of the present invention.

Include suitable for the treatment in the appropriate aspect of the present invention and/or some examples of the B cell tumour of targeting：

Chronic lymphocytic leukemia/small-sized lymphatic lymph cancer, B cell pre-lymphocytic leukemia, pouring Bar plasmacytic lymphoma is (such asMacroglobulinemia), splenic marginal zone lymthoma, plasma cell tumor (for example Plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin storage disorders, heavy chain disease), extranodal marginal zone B cell lymphoma (MALT lymthomas), knot marginal zone B-cell lymphoma (NMZL), follicular lymphoma, lymphoma mantle cell, diffusivity large B cell Lymthoma, mediastinum (thymus gland) large B cell lymphoid tumor, intravascular large B cell lymphoma, lymphoma primary effusion and Hugh Burkitt Lymthoma/leukaemia.

Being adapted in use to some examples of the tumour of the FGFR2 targeting agents treatment of the present invention includes wherein catastrophic event and tumour Those of formation and development correlation, including breast cancer, carcinoma of endometrium and stomach cancer.

Being adapted in use to some examples of the tumour of the bone marrow derived of the CD33 targeting agents treatment of the present invention includes Acute Meyloid Property leukaemia (AML).

It is adapted in use to some other realities of the tumour of PSMA (PSMA) targeting agent treatment of the present invention Example includes prostate cancer and the cancer of the brain.

Be adapted in use to the present invention human epidermal growth factor receptor-2 (HER-2) targeting agent treatment tumour it is some other Example includes breast cancer.

Being adapted in use to some other examples of the tumour of the mesothelin targeting agent of present invention treatment includes malignant tumour as between Rind gall, oophoroma, lung cancer and cancer of pancreas.

Antibody conjugates are stable during the acceptable time of storage, and this is crucial tribute to success of the invention Offer.Therefore, the stability of the drug products of on-radiation antibody conjugates and final thorium mark must is fulfilled for radiopharmaceutical Strict standard needed for product manufacturing and distribution.Having now surprisingly been found that is, the preparation as described herein card targetted comprising tissue Understand the excellent stability in storage.It is also suitable at a high temperature of the stability study for being generally used for accelerating.

One suitable for the present invention it is all compatible in terms of embodiment, tissue targeting complex compound dissolve in In suitable buffer solution.Especially, it has been found that unexpected stabilization formulations are provided using citrate buffer agent.It is preferred that lemon Hydrochlorate buffer (pH4-7) in the range of 1-100mM, particularly in the range of 10-50mM, but most preferably 20-40mM lemon Lemon hydrochlorate buffer.

Another suitable for the present invention it is all compatible in terms of embodiment, organize targeting complex compound it is solvable In the suitable buffer comprising to aminobutyric acid (PABA).Citrate buffer agent is preferably combined as (preferably herein Described in concentration under) combined with PABA.PABA for any aspect (including combination with other reagents) of the present invention Preferred concentration is about 0.005 to 5mg/ml, preferably 0.01 to 1mg/ml, more preferably 0.01 to 1mg/ml.Most preferably 0.1 to 0.5mg/ml concentration.

Another suitable for the present invention it is all compatible in terms of embodiment, organize targeting complex compound it is solvable In the suitably buffer comprising ethylenediamine tetra-acetic acid (EDTA).Preferably it is combined as using EDTA and Citrate buffer Agent.It is particularly preferred to be combined as in the presence of PABA using EDTA and citrate buffer agent.In such combination, preferred lemon Lemon hydrochlorate, PABA and EDTA suitably exist with concentration range shown in this article and preferred concentration range.For appointing for the present invention In terms of meaning the EDTA of (including combination with other reagents) preferred concentration be about 0.02 to 200mM, preferably 0.2 to 20mM and Most preferably 0.05 to 8mM.

Another suitable for the present invention it is all compatible in terms of embodiment, organize targeting complex compound it is solvable In the suitable buffer for including at least one polysorbate (sorbitan fatty acid esters of PEG grafting).It is preferred that Polysorbate includes polysorbate80 (polyoxyethylene (20) Arlacel-80), polysorbate60 (polyoxy Ethene (20) sorbitan monostearate), polysorbate40 (polyoxyethylene (20) sorbitan list palmitic acid Ester), polysorbate80 (polyoxyethylene (20) Arlacel-20) and its mixture.Polysorbate80 (P80) it is most preferred polysorbate.Poly- sorb for any aspect (including combination with other reagents) of the present invention The preferred concentration of alcohol ester (preferred polysorbate especially illustrated above) be about 0.001 to 10%w/v, preferably 0.01 to 1%w/v and most preferably 0.02 to 0.5w/v.

Although being recited as radiostabilizer (referring to US4880615A) before PABA, in the present invention, observation To positive roles of the PABA to the on-radiation conjugate of storage.In the case of in the absence of RADIATION DECOMPOSITION, the stabilization structure Into particularly surprising advantage, because the synthesis of the chelating agent of tissue targeting is generally occurred mainly in contacts it with thorium ion Before.Therefore, the chelating agent of tissue targeting can be produced being contacted with thorium ion in first 1 hour to 3 years, and preferably in this period Storage is contacted with PABA at least a portion time.That is, the step a) of the present invention and b) can be 1 hour before the step c) Carried out in 3 years and between step b) and step c), the chelating agent for organizing targeting can be contacted storage with PABA, particularly In buffer (such as citrate buffer agent), and optionally contact storage with EDTA and/or polysorbate.All material Type and concentration preferably shown in this article.Therefore, PABA is the component of highly preferred invention formulation, and can cause group Knit the long-time stability of the chelating agent of targeting and/or the thorium complex of tissue targeting.Fig. 1 illustrates PABA in the system of the present invention Effect.

Another is provided on the tissue target in the preparation of the present invention using citrate buffer agent specifically described herein To thorium complex stability unexpected advantage.The present inventor is directed to the shadow that buffer agent solution is produced to hydrogen peroxide Sound has carried out radiation research, achieves unexpected result.Known hydrogen peroxide is formed due to water RADIATION DECOMPOSITION, and is helped In the chemical modification of Proteins In Aqueous Solutions conjugate.Therefore, hydrogen peroxide is produced to have the purity and stability of product and paid no attention to The effect thought.Fig. 2 shows unexpected observation result：Compared with the every other buffer tested, in citrate In buffer, lower level peroxide is measured in the antibody HOPO conjugate solutions of the invention irradiated with Co-60 (10kGy) Change hydrogen.Therefore, preparation of the invention will preferably comprise citrate buffer agent as described herein.

In addition, present inventors have shown that another unexpected combined effect on some components in invention formulation Discovery.This again relates to the stability of radiolabeled conjugate.The purpose of research is during assessing storage²²⁷Th- The stability (see below) of AGC1118 conjugates.Under about 8000Bq/ μ g specific activity, use²²⁷Th-AGC1118 is carried out With reference to IRF experiments.Use 0.02,0.2 or 2mg/mL pABA of 30 or 100mM citrate buffer agents or addition 30mM lemons It is prepared by hydrochlorate buffer (pH 5.5)²²⁷Th-AGC1118 five kinds of different storage solutions.Fig. 3 is shown to invention formulation The significant positive role of radioactive steady, particularly when the citrate and/or PABA groups with shown scope herein During conjunction.It is maximally effective buffer that citrate is had found in the studies above, it was thus unexpectedly found that this acts through addition PABA still further improves.

The method of the present invention, the key component of complex compound and preparation are octadentate chelating agent part.For thorium ion and hydroxyl The maximally related Previous work of pyridone ligand complex has been published in WO2011/098611, and is disclosed relatively easily Produce the thorium ion with the ligand complex of the HOPO containing octadentate.

The chelating agent of previously known thorium also includes poly- amino polyacids chelating agent, and it includes the poly- of straight chain, ring-type or side chain Azepine alkyl (polyazaalkane) skeleton, acid groups (such as carboxyalkyl) are connected at the nitrogen of skeleton.This quasi-chelate compound Example includes DOTA derivatives such as to isothiocyanatobenzyl-Cyclen -1,4,7,10- tetraacethyls And DTPA derivatives are such as to isothiocyanatobenzyl-diethylene-triamine pentaacetic acid (p-SCN-Bz- (p-SCN-Bz-DOTA) DTPA), the former is cyclic chelators, and the latter is linear chelatropic agent.

The derivative of DOTA had previously been illustrated, but standard Method is not easily adapted for making thorium chelate with DOTA derivatives.The heating of DOTA derivatives and metal effectively provides chelate, but Usual yield is low.There is the tendency of at least a portion part irreversible denaturation in this process.Further, since it is to can not inversion Property relatively high neurological susceptibility, it usually needs the attachment of targeting moiety is avoided, until completing all heating stepses.Which increase The extra chemical step that must be carried out during the period of decay of transmitting α thorium isotope (has all necessary post processings And separation).Obviously, preferably α material is not launched in processing by this way, or not with degree generation greater than necessary accordingly Waste.In addition, preparing a part of thorium of waste of all times that conjugate is spent, it will decay during the preparation.

In in all respects, critical aspects of the invention are to use octadentate part, particularly comprising four HOPO parts The part of octadentate hydroxyl pyridone.This kind of part generally comprises at least four chelation groups, and it has following independently of one another Substituted pyridine structure (I)：

Wherein R¹For alkyl such as C₁To C₅The alkyl group of straight or branched, including methyl, ethyl, n-propyl or isopropyl With normal-butyl, sec-butyl, isobutyl group or the tert-butyl group.It is preferred that R¹For C₁To C₃, especially methyl.In a preferred embodiment party In case, methyl substituents are present on the nitrogen of all four parts of formula (I).

Alkyl specifically described herein is usually the C of straight or branched₁To C₈Alkyl, such as methyl, ethyl, n-propyl or different Propyl group, normal-butyl, isobutyl group, the tert-butyl group or sec-butyl etc..

In some previous disclosures, such as WO2013/167756, WO2013/167755 and WO2013/167754 In, corresponding to R¹Group be mainly solubilization radical such as hydroxyl or hydroxyalkyl (such as-CH₂OH、-CH₂-CH₂OH、-CH₂-CH₂- CH₂OH etc.).This has certain advantage in terms of higher dissolubility, but is due to R¹Reactivity on position, this quasi-chelate compound It is difficult with amido link and is connected to targeting moiety.Therefore, in the present invention, R¹Generally it is not hydroxyl or hydroxyalkyl.

In formula (I), group R²To R⁶H, OH ,=O, coupling moiety and junction portion can be each independently selected from.It is preferred that Ground, group R²To R⁶In just have one for=O, and group R²To R⁶In just have one be OH.Group R²To R⁶Middle residue Three can be H, but R²To R⁶In at least one be junction portion and/or coupling moiety.Coupling moiety is described below, but Its end is that carboxylic acid is used to be connected with targeting moiety by amido link.This kind of coupling moiety may be connected directly in group R²To R⁶ One of on the ring at place, but be more preferably connected to junction portion, its own will constitute group R²To R⁶One of.

3, the 2-HOPO parts of N- substitutions are very preferably as the HOPO groups of the present invention, and in an embodiment In, all four complexing moieties of octadentate part can be 3,2-HOPO parts.

Suitable chelating moiety can be formed by methods known in the art, including be recorded in US5, and 624,901 is (such as real Apply example 1 and 2) and WO2008/063721 (the two is included in the application by reference) in method.

It is preferred that chelation group include with following formula (II) those：

In above-mentioned formula (II) ,=O parts represent the oxo group being connected on any carbon of pyridine ring, and-OH, which is represented, to be connected It is connected to hydroxylic moiety and-R on any carbon of pyridine ring_LRepresent by Hydroxypyridinone be partially attached to other complexing moieties with Form the junction portion of whole octadentate part.Any junction portion specifically described herein is suitable as R_L, including short alkyl such as C₁Extremely C₈Alkyl, including C₁To C₈Alkyl, alkenyl or alkynyl, including the methyl of all topological structures, ethyl, propyl group, butyl, amyl group and/ Or hexyl.R_LFormula (II) ring can be connected at any carbon of pyridine ring.Then R_LGroup can be bound directly to another chelating Partly, another linking group and/or central atom or group such as ring or other templates (as described herein).Selection joint, Chelating moiety and optional template part are to form suitable octadentate part.

R_cCoupling moiety is represented, as described below.Suitable part include end for the alkyl such as alkyl of hydroxy-acid group or Alkenyl.Present inventor have determined that, such as by the method for the present invention, acid amides is formed using carboxylic acid coupling part can be in chelating agent More stable be conjugated is provided between the part of tissue targeting.

In a preferred embodiment ,-the OH of Formula II and=O parts are located on the adjacent atom of pyridine ring, therefore 2,3-, 3,2-, 4,3- and 3,4- pyridone ketone derivatives are all most suitable.Group R_NFor methyl substituents.

In a preferred embodiment, four 3,2 hydroxy pyrimidine ketone part is present in octadentate ligand structure.

Preferred chelation group is those of formula (IIa)：

As used herein, term " the junction portion " (R in formula (II) and formula (IIa)_L) be used to represent to be used to connect eight The chemical entities of at least two chelation groups in tooth part, the octadentate part forms crucial group in various aspects of the invention Point.Junction portion is also connected to the coupling moiety of the part for octadentate ligand moiety to be coupled to tissue targeting.Generally, often Individual chelation group (such as those of above-mentioned formula (I) and/or (II) and/or (IIa)) is by for bidentate, therefore four HOPO chelatings Group there typically will be in part.This kind of chelation group is connected to each other by their junction portion, and passes through coupling moiety It is coupled to the part (in the method for the invention) of tissue targeting.Therefore, junction portion (such as group R of formula (II)_L) can be Shared between formula (I) and/or (II) chelation group more than one.Junction portion also act as octadentate part complexing moiety and Tie point between targeting moiety.In this case, at least one junction portion will be connected to coupling moiety (in formula (II) R_C).Suitable junction portion includes short alkyl such as C₁To C₁₂Alkyl, including C₁To C₁₂Alkyl, alkenyl or alkynyl, including all open up Flutter methyl, ethyl, propyl group, butyl, amyl group and/or the hexyl of structure.It can be included in junction portion (R_L) in other groups bag Include any functional group's such as aryl (such as phenyl), acid amides, amine (especially secondary amine or tertiary amine) and/or ether suitably stablized.R_C Part can also include alkyl and/or aryl moieties and optional group such as amine, acid amides and ehter bond.Generally, coupling moiety is all Component will need stable under the condition of storage that complex compound is experienced by.This is decomposed including alpha radiation, therefore preferably not unstable Functional group.

In one embodiment, coupling moiety includes carboxylic acid, at least one moieties (such as methyl or second of end Base section), at least one acid amides and at least one aryl moiety (such as phenyl).Coupling moiety can by carbon-carbon bond, acid amides, Amine and/or ehter bond are connected to one or more junction portions of octadentate part.

In the most preferred embodiment of the present invention, octadentate part is connected to the coupling moiety (R of targeting moiety_C) choosing From

[-CH₂- Ph-N (H)-C (=O)-CH₂-CH₂- C (=O) OH],

[-CH₂-CH₂- N (H)-C (=O)-(CH₂-CH₂-O)_1-3-CH₂-CH₂- C (=O) OH] or

[-[CH₂]_1-3- Ar-N (H)-C (=O)-[CH₂]_1-5- C (=O) OH], wherein Ar is for aromatic group such as substitution or not Substituted phenylene, and Ph is phenylene, preferred pair phenylene.

Junction portion can be or comprising any other suitable stable chemical bond, including ester, ether, amine and/or amide groups.It is logical The total atom number (if more than a paths, then being counted by shortest path) of two chelating moieties of often limitation connection, with Just chelating moiety is limited in and formed for complex compound in suitable arrangement.Therefore, junction portion is generally selected with chelating portion No more than 15 atoms an of/offer, provide 1 to 12 atom, more preferably 1 to 10 atom preferably between chelating moiety. When junction portion is directly connected to two chelating moieties, the length of joint is usually 1 to 12 atom, preferably 2 to 10 (such as second Base, propyl group, normal-butyl etc.).When junction portion is connected to center die plates (see below), then each joint can be shorter, two Single joint connection chelating moiety.In the present case, preferably joint length is 1 to 8 atom, preferably 1 to 6 atom (methyl, Ethyl and propyl group are suitable, and group is as at one end or two ends have ester, ether or amido link).

The joint for being connected to each other and/or being connected with center die plates except the various chelation groups being mainly used in octadentate part Outside part, octadentate part also includes the coupling moiety (R with terminal carboxylic_C).The function of coupling moiety is by stable Octadentate part is connected to targeting moiety by covalent bond --- especially acid amides ---.Preferably, coupling moiety will be by directly common Be connected in chelation group one of valency more often by being connected to junction portion or template is covalently attached to chelating Group.If using more than two coupling moieties, each coupling moiety may be connected to arbitrarily available site, such as arbitrary mould Arbitrarily available site on plate, joint or chelation group.

In one embodiment, coupling moiety can have following structure：

Wherein R⁷For bridging part, it is selected from substituted or unsubstituted alkyl, substituted or unsubstituted miscellaneous alkyl, substitution Or the member of unsubstituted Heterocyclylalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl；X is to pass through acid amides Or carboxylic acid or the targeting moiety of functional group's connection of equal value.It is preferred that bridging part include it is all shown in this article as suitable Those groups of junction portion.

It is preferred that targeting moiety include those all specifically described herein.And it is preferred that reactive X group include energy Enough any groups with being acted in targeting moiety formation acid amides covalent bond as " carboxylic acid ", including such as-COOH ,-SH ,-NHR And group, wherein NHR R can be any short alkyl of H or as described herein.It is connected to the highly preferred group on targeting moiety ε-amine including lysine residue.The non-limiting examples of suitable reactivity X group include n-hydroxysuccinimide base ester, Imide base ester, carboxylic acid halides, N- maleimides and alpha-halogen acetyl group.

In a preferred embodiment in accordance with this invention, selection bridging part R⁷For substituted aryl, and select by Octadentate part is connected to the coupling moiety (R of targeting moiety_C) it is [- C (=O)-CH₂CH₂- X-], it is thus free on HOPO parts Carboxylate group is in situ living in the form of N-hydroxy-succinamide ester in aqueous immediately before conjugated with targeting moiety Change.

Coupling moiety is preferably connected so that the coupling octadentate part of gained is possible to form stable complexing of metal ion Thing.Therefore, coupling moiety will preferably be connected to joint, template or chelating moiety at the site for not interfering significantly with complexing.It is this kind of Site will be preferably on joint or template, more preferably at the position away from the surface combined with target.

Formula (I) or (II) or (IIa) each part can be by specifically described herein any suitable in octadentate part Linking group and the remainder that part is connected to suitable topological structure.For example, four formulas (I) or (II) or (IIa) Group is connected on skeleton to form linear ligands by its linking group, or can be bridged by linking group " low to be formed Polymers " type structure (it is linear or ring-type).Or, formula (I) and/or (II) and/or (IIa) ligand moiety can each lead to Cross joint (such as " R_L" part) central atom or group are connected to " intersection " or " star " form.Joint (R_L) partly can be single Solely connected by carbon-carbon bond；Or can be connected to each other by any moderately stable function (including amine, acid amides, ether or thioether bond), Other chelation groups are connected to, are connected on skeleton, template, coupling moiety or other joints.

" star " arrangement is represented with hereafter formula (III)：

Wherein all groups and position are as it appears from the above, in addition, atom or template group, such as carbon atom, alkyl centered on " T " Chain (such as herein-above set forth any those), aliphatic ring or aromatic ring (including heterocycle) or fused ring system.Most basic template is Single carbon, it can then be connected through group and be connected to each chelating moiety.Longer chain such as ethyl or propyl group are equally may be used Capable, two of which chelating moiety is connected to each end of template.Obviously, any suitable stable keys can be used for connection template and connect Head point, the stable keys include carbon-carbon bond, ester, ether, amine, acid amides, thioether or disulfide bond.

Obviously, in formula (II), (III), (IV) and (IVb) structure, (joint or coupling portion are not passed through for example originally Point) substitution pyridine ring those positions can --- if appropriate --- with R in formula (I)¹To R⁵Described substituent.Especially Ground, small alkyl substituent (such as methyl, ethyl or propyl group) may be present in any position.

Octadentate part will extraly include at least one coupling moiety as described above.This can be to include shown in this article Anticipate any suitable structure of those, and blocked in final complex compound or carboxylic acid in the method for the invention with targeting moiety.

Coupling moiety may connect to any suitable site of joint, template or chelating moiety, such as shown in formula (III) A, b and/or c site.The connection of coupling moiety can by any suitably stable key, for example carbon-carbon bond, ester, ether, amine, Acid amides, thioether or disulfide bond.Analogously it is possible to which the group for forming any this key with targeting moiety is applied to the official of coupling moiety Energy end, and the part will be with this kind of group end capping when being connected to targeting moiety.

A kind of " skeleton " type structure of replacement is shown in in following formula (VI)

Wherein all groups and position are as noted above, and " R in addition_B" be skeleton part, its generally with institute herein Any junction portion shown has similar 26S Proteasome Structure and Function, therefore can permit any definition of junction portion applied to context Perhaps skeleton part.Suitable skeleton part will form support, and wherein chelating moiety is connected to the support by its linking group On.It is generally necessary to three or four skeleton parts.Typically for linear backbone, it is three, if skeleton is cyclized, for Four.Particularly preferred skeleton, which is partly comprised in one or both ends, optionally has hetero atom or the short hydrocarbon chain (example of functional moiety As described herein those).In this regard, amine and amide groups are particularly suitable for.

Coupling moiety may connect to any suitable site of joint, skeleton or chelating moiety, such as shown in formula (IV) A, b and/or c' site.The connection of coupling moiety can by any suitably stable key, for example carbon-carbon bond, ester, ether, amine, Acid amides, thioether or disulfide bond.Analogously it is possible to which the group for forming any this key with targeting moiety is applied to the official of coupling moiety Energy end, and the part will be with this kind of group end capping when being connected to targeting moiety.

" skeleton " type octadentate with four 3,2-HOPO chelating moieties being connected to by amide linker group on skeleton The example of part is with following formula (V)：

Obviously, coupling moiety R_CAny suitable site that can be on the molecule is added, such as at a secondary amine group Or added at the branch point on any skeleton alkyl.Group R_CPreferred site be shown in formula (V).In the suitable of the present invention Aspect, R_CUsing carboxylic acid as end, or by amido link be connected to tissue target part.All small alkyl group such as skeletons Propylidene or the ethylidene of n- substitutions can be by for example any those arbitrary (wherein, Asias specifically described herein of other small alkylidenes Methyl, ethylidene, propylidene and butylidene are fit closely) substitution.

Each there are four 3,2-HOPO chelating moieties that ethylenediamine and propane diamine are respectively connecting to by acetamide group Exemplary " templating " octadentate part be with following formula (VI)：

Obviously, any alkylidene group for being shown in formula (VI) as ethylene moieties can be independently by other small alkylidenes Group such as methylene, propylidene and the substitution of positive butylidene.Advantageously, symmetry, therefore preferred center propylidene C are remained₃ Chain and remaining ethylidene retain, or two ethylidene that HOPO is partially attached on one or two center tertiary amine can quilt Methylene or propylidene are replaced.

Formula (VIb) shows coupling moiety R_CPossibility site, the R_CFormula (VI) will be present in any suitable position In, such as-CH- groups.

As it appears from the above, octadentate part will generally include to be connected to the coupling portion of the remainder of part in any site Point.The suitable site of coupling moiety connection is shown in in following formula (VIb)：

Wherein R_CFor any suitable coupling moiety, the particularly idol for being connected to the group that tissue is targetted via amido Join part.It is acid or the short alkyl such as C of active group of equal value to form the end of acid amides for the part with tissue targeting₁To C₈ The aromatics or aliphatic group of ring-type, side chain or straight chain are highly suitable as the group R in formula (VIb) and full text_C。

Exemplary template also includes wherein coupling group R_CIt is covalently attached to other on the nitrogen-atoms in amino skeletal Template, as shown in formula (VII).

Showing the highly preferred octadentate part in the suitable site of part connection is included with following formula (VIII) and (IX) Those：

The synthesis of compound (VIII) is described below and follows synthetic route described below.

There is end to be carboxylic acid group for the compound formation of AGC0019 and formula (VI), (VIb), (VII), (VIII) and (IX) The preferred octadentate chelating agent of the junction portion of group.Octadentate part and shown junction portion also shape shown in those structures Into their type preferred embodiment and can be combined with any combinations.This kind of combination is obvious to technical staff.

The step a) of the method for the present invention can be carried out by any suitable synthetic route.It is generally included by linker Four HOPO parts (such as formula (I) and/or (II) and/or (IIa)) are connected to coupling moiety by group, optionally by mould Plate is carried out.All these groups are described herein, and preferred embodiment is equally preferred in this context. Coupling between HOPO parts, joint, coupling moiety and optional template generally using stable group such as acid amides, amine, ether or carbon- Carbon key.The method and any required Preservation tactics for synthesizing this generic key are well known in synthesis chemical field.Synthetic method Provided in the following embodiment of some specific examples below.This kind of method provides specific example, but shown in it Synthetic method can also be used by those skilled in the art under general background.Therefore, the method shown in embodiment is also aimed to It is used as general disclosure, it is adaptable to all aspects of the invention and embodiment that context allows.

Preferably, in all aspects of the invention, the complex compound of the thorium and octadentate part of launching α need not be heated to 60 DEG C More than (need not for example be heated to more than 50 DEG C), preferably without being heated to more than 38 DEG C and need not most preferably be heated to more than 25 DEG C (such as in the range of 20 to 38 DEG C) and formed or can be formed.Conventional scope can be, such as 15 to 50 DEG C or 20 to 40 DEG C. Complex reaction (part c)) in the method for the present invention can be carried out persistently any rational period, but it is preferably 1-120 points Clock, preferably 1-60 minute, more preferably 5-30 minute.

Also it is preferred that after preparing the conjugate of targeting moiety and octadentate part addition transmitting α thorium isotope (example Such as²²⁷Th⁴⁺Ion).It is therefore preferable that by using octadentate part and the conjugate (chelating of tissue targeting of the part of tissue targeting Agent) it is complexed transmitting α thorium isotope (for example²²⁷Th⁴⁺Ion) and formed or the product of the present invention can be formed.

Different types of target compound can be connected to via octadentate chelating agent (including coupling moiety specifically described herein) Thorium (such as thorium -227).Targeting moiety may be selected from known targeting group, and it includes monoclonal antibody or polyclonal antibody, growth The factor, peptide, hormone and hormone analogs, folic acid derivatives, biotin, avidin and Streptavidin or its is similar Thing.RNA, DNA or its fragment (such as fit) of other possible targeting groups including suitable functionalization, oligonucleotide, Carbohydrate, lipid pass through compound for preparing these groups with or without protein combination etc..As described above, can Including peg moiety, thus to increase biological resident time and/or reduce immunostimulation.

Generally, as used in this article, the part of tissue targeting is " peptide " or " protein ", and it is to be main by amino acid group / amide backbone formation structure, with or without two grades and tertiary structure attributes.

In one embodiment, tissue targeting moiety can exclude the conjugated antibody of bone material, liposome and folic acid or Antibody fragment.

According to the present invention,²²⁷Th can by will by amido link connection or attachable complexing agent targeted to as described herein group The part of targeting is knitted to be complexed.Generally, the molecular weight of targeting moiety is that 100g/mol to millions of g/mol (is particularly 100g/mol to 1,000,000 g/mol), and the related acceptor of preferred pair disease directly has compatibility, and/or comprising suitable The bonding agent (such as biotin or avidin) given in advance, the bonding agent is being given²²⁷Be bound to before Th targeted to The molecule of disease.Suitable targeting moiety includes polypeptide or oligopeptide, protein, DNA and RNA fragments, fit etc., optimization protein (including IgG and IgM types are anti-for matter, such as avidin, streptavidin, polyclonal antibody or monoclonal antibody Body), or the fragment or construct of protein or protein mixture.Particularly preferred antibody, antibody construct, antibody fragment (such as Fab fragments or any fragment comprising at least one antigen binding regions), fragment construct (such as single-chain antibody) or Its mixture.Suitable fragment particularly including Fab, F (ab')₂, Fab' and/or scFv.Antibody construct can be shown in herein Any antibody or fragment.

The first targeting embodiment suitable for all aspects of the invention, specific-binding agent (tissue may be selected The part of targeting) target CD22 acceptors.The part of this kind of tissue targeting can be to have with least one sequence set forth below The peptide of sequence similarity or homogeneity：

Light chain：

Heavy chain：

In above-mentioned sequence, "-" represents the unchanged residue from mouse sequence in humanization (H'ised) sequence.

In above-mentioned sequence (SeqID1-5), runic region is considered as crucial specific binding area (CDR), lower stroke Line region is considered as in combination being secondary important, and the region do not emphasized is considered as representative structure region rather than spy Different in nature calmodulin binding domain CaM.

In all aspects of the invention, organize targeting part can have with those sequences listed in SeqID1-5 At least one or any one there is the sequence of basic sequence identity or basic sequence similarity.Basic sequence is same One property/similitude is considered to have with complete sequence at least 80% and/or (runic shows in above-mentioned sequence with specific binding region Those regions shown and those parts optionally underlined) at least 90% sequence similarity/homogeneity.For runic Region and still more preferably for complete sequence, sequence similarity or preferred homogeneity preferably can be at least 92%, 95%th, 97%, 98% or 99%.The Genetics from University of Wisconsin can be used in sequence similarity and/or homogeneity " BestFit " programs of the software kits of Computer Group Version 10 is determined.The program, which is used, has following default value Smith and Waterman local algorithm：Gap creation penalty=8, gap extension penalties=2, Mean match=2.912, Average Mismatch 2.003.

The part of tissue targeting can include more than one peptide sequence, wherein at least one sequence and preferably all of sequence It can (independently) meet the above-mentioned sequence similarity and preferred sequence identity with any of SeqID1-5.

The part of tissue targeting can have binding affinity for CD22, and in one embodiment, it can also phase There is the sequence at most of about 40 variations (preferably 0 to 30 variation) for complete domain.Variant can pass through insertion, missing And/or displacement is formed, and can be continuous or discrete for SeqID1-5.Displacement or insertion generally pass through heredity At least one progress in 20 amino acid of password, and displacement most typically be conservative substitution.

The second targeting embodiment suitable for all aspects of the invention, specific-binding agent (tissue may be selected The part of targeting) target CD33 acceptors.The part of this kind of tissue targeting can be monoclonal antibody, and it is that woods is appropriate that it, which may be selected, Pearl monoclonal antibody (lintuzumab) has the lintuzumab of extra lysine residue in C-terminal.

The 3rd targeting embodiment suitable for all aspects of the invention, specific-binding agent (tissue may be selected The part of targeting) target HER-2 antigens.The part of tissue targeting can be monoclonal antibody and preferably Herceptin (trastuzumab)。

Other suitable antibody sequences for targetting FGFR2, mesothelin and PSMA are illustrated in embodiment part.However, To those skilled in the art it should be obvious that the known targeting disease comprising lysine residue is special in the sequence Any protein form of specific target correspondingly applies to every other aspect by for the alternatives of the inventive method.

For launching α thorium component, nearest key is the discovery that, some alpha activity thorium isotopes are (for example²²⁷Th) It can be administered with the amount that treats effectively and will not produce the bone marrow toxicity that can not be resistant to.In all aspects of the invention, thorium- 227(²²⁷Th) it is preferred thorium isotope.As used in this article, term " acceptable non-bone marrow toxicity " is used to represent, most Importantly, the amount of the radium -223 produced by the radioisotopic decay of the thorium -227 given is typically not enough to make subject It is directly lethal.However, for it is obvious to the skilled person that bone marrow injury amount (and possibility of lethal response) --- it is The acceptable side effect of this treatment --- by with the prognosis of the type of disease to be treated, the target of therapeutic scheme and subject And significant changes.Although the preferred subject of the present invention is the mankind, other mammals, particularly companion animals such as dog, The use of the present invention is will benefit from, and the level of acceptable bone marrow injury can also reflect the species of subject.It is acceptable The level of bone marrow injury is bigger than in treatment nonmalignant disease generally in treatment malignant disease.One well-known marrow Toxic level is measured for neutrophil count, and in the present invention,²²³The amount of Ra acceptable non-bone marrow toxicity Typically controlled amount so that the neutrophil cell fraction of (minimum point) is not less than what is counted before treatment at its minimum point 10%.Preferably,²²³The amount of Ra acceptable non-bone marrow toxicity is so that neutrophil cell fraction is at least in minimum point 20% and more preferably at least 30% amount.Most preferably at least 40% minimum point neutrophil cell fraction.

In addition, comprising radiothorium (for example²²⁷Th compound) can be used for high dose scheme, wherein when including stem cell When support or suitable restoration methods, produced radium is (for example²²³Ra bone marrow toxicity) is typically not tolerable.This kind of In the case of, neutrophil count decreases below 10% in minimum point, and especially will decrease to 5% or if need To be less than 5%, condition is to take appropriate precautionary measures and then give stem cell to support.These technologies are ripe in the art Know.

Thorium isotope of special interest is thorium -227 in the present invention, and thorium -227 is under where the context permits The preferred isotope of all thoriums referred to herein.Thorium -227 is relatively easily produced, and can be indirectly by by neutron exposure²²⁶Prepared by Ra, it will include²²⁷Th parent nucleus, i.e.,²²⁷Ac(T_1/2=22 years).Actinium -227 can easily with²²⁶Ra targets Separation, and be used as²²⁷Th generator (generator).If desired, this method can be amplified to commercial scale, therefore it can keep away Exempt from the supply problem for being considered as occurring in other most of alpha emitters of the alternatives of molecular targeted radiotherapy.

Thorium -227 decays via radium -223.In this case, the half-life period of primary daughter is 11.4 days.By pure²²⁷Th Source, the radium of moderate was only produced at initially several days.However,²²³Ra genotoxic potential is higher than²²⁷Th genotoxic potential, because coming From α particles²²³Be exactly within a few minutes after Ra transmitting from short life daughter three kinds of other α particles (referring to table 2 below, Wherein list the decay series of thorium -227).

Table 2

Partially due to it produces potentially harmful the decay constant, (T of thorium -227_1/2=18.7 days) not yet it is broadly contemplated use In α particle therapies.

In order to distinguish most abundant naturally occurring thorium isotope, (i.e. (half-life period is 1010 to thorium -232, and is effectively non- Radioactivity)) thorium complex, it should be appreciated that thorium complex claimed and combinations thereof includes being more than natural relative herein ((i.e. half-life period is less than at least one of the thorium of 103 years together to the transmitting α of for example, at least big 20%) thorium radioisotope to abundance Position element, such as thorium -227).The demand does not influence the definition of the inventive method, wherein being distinctly claimed the radioactivity of therapeutically effective amount Thorium such as thorium -227, but be preferably all such case in all respects.

In all aspects of present aspect, preferred emission α thorium ion is the ion of thorium -227.The 4+ ions of thorium be for The preferred ion of the complex compound of the present invention.Correspondingly, the 4+ ions of highly preferred thorium -227.

Thorium -227 is not tolerable to cause without producing so many radium -223 to be enough the therapeutic effect needed for providing The amount of bone marrow suppression is given.Expect to maintain daughter isotope in target area to obtain further treatment effect by its decay Really.However, it is not necessary to maintain the control of thorium decay constant so as to useful therapeutic effect without causing unacceptable bone Marrow toxicity.

Assuming that tumour cell kills effect essentially from thorium -227 rather than from its daughter, then the isotope is possible Therapeutic dose can be determined by being compared with other alpha emitters.For example, for astatine -211, the therapeutic dose in animal is usual For 2-10MBq/kg.By correcting half-life period and energy, the corresponding dosage of thorium -227 is at least 36-200kBq/kg body weight.This will It is right²²⁷Th amount setting lower limit, the lower limit can effectively be given, expect to obtain therapeutic effect.The guarantor of the calculation assumption astatine and thorium Allowance is suitable.It is obvious that ground, 18.7 day half-life period of thorium most likely results in the bigger elimination of the isotope before its decay. Therefore, the dosage of the calculating is generally considered to be the effective dose of minimum.With what is be fully retained²²⁷Th is (i.e. not from internal elimination 's²²⁷Th) therapeutic dose represented is generally at least 18 or 25kBq/kg, preferably at least 36kBq/kg and more preferably at least 75kBq/kg, such as 100kBq/kg or bigger.It is expected that a greater amount of thoriums is by with bigger therapeutic effect, but if producing not Tolerable side effect, then can not give.Similarly, if with short biological half-life (i.e. from the body for still carrying thorium Body eliminate before half-life period) form give thorium, then need a greater amount of radio isotopes to realize therapeutic effect, because It will be eliminated before its decay for a large amount of thoriums.However, the amount of produced radium -223 will be reduced accordingly.When isotope is fully retained When, the dosage that the above-mentioned amount of thorium -227 to be administrated can be easily equal to what it is with shorter biological half-life is related.This kind of meter It is well known in the art at last, and provided in WO04/091668 (such as in Examples 1 and 2).

If radiolabeled compound releases daughter nucleic, knows the final result of any radioactive daughter nuclide (if applicable) it is important.For²²⁷Th, main daughter products are²²³Ra, due to its bone characteristic that becomes, it is in clinical evaluation In.Radium -223 quickly removes blood, and be enriched in bone or discharged by the approach of intestines and kidney (referring to Larsen,J Nucl Med 43(5,Supplement):160P(2002)).Therefore, by²²⁷The radium -223 that Th discharges in vivo Will not largely unhealthful soft tissue.In M ü ller Int.J.Radiat.Biol.20:233-243(1971) In on the citrate as dissolving²²⁷In the research of Th distribution, find in soft tissue by²²⁷What Th was produced²²³Ra holds Change places and redistribute in bone or excrete.Therefore, the known toxicity of transmitting α radium, the particularly known toxicity to marrow, be The problem of thorium dosage.

Confirm first in WO 04/091668, in fact, can be given in human experimenter and be resistant at least 200kBq/ Kg's²²³Ra dosage.These data are provided in this publication.Therefore, it can be seen that at present, very it is surprising that really In the presence for the treatment of window phase, wherein can be by therapeutically effective amount²²⁷Th (being greater than 36kBq/kg) gives mammalian subject, And do not expect unacceptable risk of this kind of subject by serious or even lethal bone marrow toxicity.Nevertheless, best Ground is of crucial importance using the treatment window phase, it is thereby necessary that radiothorium is quickly and efficiently complexed and with very high Affinity is kept, so that the maximum possible ratio of dosage is delivered to target site.

By²²⁷What Th medicines were produced²²³Ra amount is by depending on the biological half-life of radiolabeled compound.Preferably Situation is that the tumour using the complex compound absorbed with Rapid tumor, including in internalization to tumour cell, strong retains and normal Short biological half-life in tissue.As long as however,²²³Ra dose maintenance is in tolerable level, then with less than preferable The complex compound of biological half-life can be useful.The amount of the radium -223 produced in vivo is by the amount of thorium to give and thorium complex The factor of biological resident time.Under any particular case, the amount of the radium -223 of generation can easily be counted by those of ordinary skill Calculate.²²⁷Th maximum dosage will determine by the amount of the radium produced in vivo, and have to be lower than that will produce can not tolerance level The amount of side effect especially bone marrow toxicity.The amount will typically be less than 300kBq/kg, especially less than 200kBq/kg and more preferably Less than 170kBq/kg (such as less than 130kBq/kg).Minimum effective dose by by the cytotoxicity of thorium, pathological tissues to production The neurological susceptibility and thorium of raw α irradiations by target complex compound (being in the present case the combination of part and targeting moiety) efficient combination, Retain and the degree of delivering is determined.

In the method for the invention, thorium complex is ideally given with 18 to the dosage of thorium -227 of 400kBq/kg body weight, It is preferred that 36 to 200kBq/kg (such as 50 to 200kBq/kg), more preferably 75 to 170kBq/kg, especially 100 to 130kBq/ kg.Correspondingly, single dosage until may include be about multiplied by suitable body weight (such as 30 to 150Kg, preferably 40 to 100Kg) this Any scope in a little scopes (such as the scope per dosage is 540kBq to 4000KBq).In addition, the dosage of thorium, complexing agent And the dosage for ideally causing the radium -223 generated in vivo is less than 300kBq/kg by method of administration, more preferably less than 200kBq/kg, still more preferably below 150kBq/kg, especially less than 100kBq/kg.Again, this will be provided by using shown Pair that any body weight is multiplied by these scopes to represent²²³Ra exposed amount.Above-mentioned dosage level is preferably²²⁷Th's is fully retained agent Amount, but in view of²²⁷Th will be before decay from internal removing, and it is alternatively dosage.

Compared with physical half time,²²⁷The biological half-life of Th complex compounds is short (such as less than 7 days, especially less than 3 days), Significantly bigger dosage is needed to provide equivalent retained dose.Thus, for example, 150kBq/kg's is fully retained dosage phase When in the half-life period given using 711kBq/kg dosage as the complex compound of 5 days., can be by using method well known in the art The biological clearance rate of complex compound calculates the equivalent dosage of any suitable retained dose.

Due to one²²⁷Th nuclear decay provides one²²³Ra atoms,²²⁷Th reservation and therapeutic activity will be subjected to patient 's²²³Ra dosage is directly related.It is any it is special in the case of produce²²³Well known method can be used to calculate for Ra amount.

In a preferred embodiment, this invention therefore provides treatment mammalian subject (such as institute herein State) in disease method, methods described include to the subject give therapeutically effective amount as described herein at least A kind of thorium complex for organizing to target.

Have clearly a need for making subject couple²²³The exposure minimum of Ra daughter isotopes, unless²²³The property of Ra daughter isotopes It is used effectively.Specifically, the amount of the radium -223 produced in vivo will be normally higher than 40kBq/kg, such as higher than 60kBq/Kg. In some cases, produce in vivo²²³Ra is necessarily higher than 80kBq/kg, such as higher than 100 or 115kBq/kg.

The complex compound that thorium -227 in suitable carrier solution is marked can pass through intravenous, intracavitary (such as intraperitoneal), skin Under, oral or local administration, given as single application or with gradation application scheme.Preferably, it is conjugated to the network of targeting moiety Compound will be administered as solution by parenteral (such as percutaneous) approach, be administered especially by intravenous or intracavitary route.It is excellent Selection of land, composition of the invention will be configured to sterile solution for parenteral.

The present invention method and product in thorium -227 can be used alone or be applied in combination with other treatment form, it is described its His form of therapy includes surgical operation, external beam radiation treatment, chemotherapy, other radionuclides or tissue temperature regulation etc..This The further preferred embodiment of the inventive method is formd, and preparation/medicine can be correspondingly comprising at least one extra For example another radioreagent of therapeutically active agent or chemotherapeutics.

In an especially preferred embodiment, subject has also carried out stem-cell therapy and/or other supporting treatments To reduce the bone marrow toxicity effect of the induction of radium -223.

The molecule of thorium (such as thorium -227) mark of the present invention can be used for treating carcinous by targeting disease associated receptor Or non-cancerous disease.Generally,²²⁷Th such medical application will by based on pass through chelating agent will²²⁷Th is connected to antibody, resisted The radioimmunotherapy of body fragment or antibody construct or antibody fragment constructs is used to treat carcinous or non-cancerous disease. In the method according to the invention and medicine²²⁷Th purposes is particularly suitable for treating any type of cancer, including cancer, sarcoma, lymph Knurl and leukaemia, especially lung cancer, breast cancer, prostate cancer, carcinoma of urinary bladder, kidney, stomach cancer, cancer of pancreas, cancer of the esophagus, the cancer of the brain, ovum Nest cancer, uterine cancer, carcinoma of mouth, colorectal cancer, melanoma, Huppert's disease and NHL.

In another embodiment of the present invention, while the patient with soft tissue and skeletal diseases can be by giving thorium And utilize²²⁷Th and in vivo generate²²³Ra is treated.In terms of this is particularly advantageous, extra therapeutic group for the treatment of Divide from the acceptable non-bone marrow toxicity amount by targetting skeletal diseases²²³Ra.In the treatment method,²²⁷Th is usual The primary and/or metastatic cancer for being used to treat soft tissue by its appropriate targeting, and by²²⁷Produced by Th decays 's²²³Ra is used for the related skeletal diseases for treating same patient.The skeletal diseases can be to cause it by primary soft-tissue cancers To the metastatic tumor of bone, or can be primary disease, wherein soft tissue therapy is confrontation metastatic cancer.Sometimes, soft tissue and Skeletal diseases can be uncorrelated (for example, additional procedures have the skeletal diseases of the patient of rheumatological soft-tissue disease).

The patient's condition for the treatment of is particularly suitable in the method for the present invention, purposes and in terms of other includes tumprigenicity and Hypertrophic disease Disease, such as cancer, sarcoma, myeloma, leukaemia, lymthoma or mixed type cancer, it includes NHL or B cell is swollen Knurl, breast cancer, carcinoma of endometrium, stomach cancer, acute myelogenous leukemia, prostate cancer or the cancer of the brain, celiothelioma, oophoroma, lung cancer Or cancer of pancreas.

The synthesis of some examples is provided below.The step of shown in these synthesis, is by suitable for many embodiment party of the present invention Case.For example, step a) can be entered by intermediate A GC0021 shown below in as described herein many or all embodiments OK.

Synthesize AGC0020 key intermediates

N, N, N', N'- tetra- (2- amino-ethyls) -2- (4- nitrobenzyls) propane -1,3- diamines

A) dimethyl malenate, sodium hydride, THF；B) DIBAL-H, THF；c)MsCl,NEt₃,CH₂Cl₂；

D) imidazoles, Boc2O, CH₂Cl₂, toluene；E) DIPEA, acetonitrile；F) MeOH, water, AcCl

Synthesize AGC0021 key intermediates

3- (benzyloxy) -1- methyl -4- [(thio -1,3- thiazolidines -3- bases of 2-) carbonyl] pyridine -2 (1H) -one

A) diethy-aceto oxalate, potassium ethoxide, toluene, EtOH；B) Pd/C, paraxylene；C) Mel, K₂CO₃, DMSO, acetone；

d)i)BBr₃, DCM, ii) and BnBr, K₂CO₃, KI, acetone；E) NaOH, water, MeOH；f)DCC, DMAP, DCM

Synthesize formula (VIII) compound chelate 4- [4- (and 3- [it is double (2- [(3- hydroxyl -1- methyl -2- oxo -1, 2- dihydropyridine -4- bases) carbonyl] amino } ethyl) amino] -2- { [double (2- { [(3- hydroxyl -1- methyl -2- oxos -1,2- bis- Pyridinium hydroxide -4- bases) carbonyl] amino } ethyl) amino] methyl } propyl group) phenyl] amino } -4- ketobutyric acids

In the method for forming the complex compound of the present invention, the coupling preferably between the part of octadentate chelating agent and tissue targeting Reaction is carried out in aqueous.This has several advantages.First, it eliminates manufacturer and removes all solvents to less than acceptable Level and the burden for proving the removing.Second, it reduce waste and above all it by avoiding separating or removing step Suddenly production is accelerated.In the context of the radiopharmaceutical of the present invention, progress synthesis as quick as possible is important, because putting Injectivity isotope will decay in all times, and the time spent in preparation wastes valuable material and introduces dirt Contaminate thing daughter isotope.

The suitable aqueous solution includes any of many buffers of purified water and buffer as known in the art.Second Hydrochlorate, citrate, phosphate (such as PBS) and sulfonic acid salt buffer agent (such as MES) are the conventional of well known aqueous buffer Example.

In one embodiment, this method includes being formed part (such as herein in the whole text institute of the octadentate comprising Hydroxypyridinone State) first aqueous solution and tissue targeting part (as described in herein in the whole text) second aqueous solution, it is and water-soluble by described first Liquid and second aqueous solution contact.

Suitable coupling moiety is hereinbefore discussed in detail, and being begged for herein as coupling and/or linking group All groups of opinion and part can be suitably used for targeting moiety being coupled on part.Some preferred coupling groups include acyl Amine, ester, ether and amine coupling group.Ester and acid amides can be by being easily formed by carboxylic acid generation activation ester group.This kind of carboxylic acid can It is present in targeting moiety, coupling moiety and/or ligand moiety, and is generally reacted with alcohol or amine to form ester or acid amides.It is this kind of Method is in the art very well known, and using well known activating reagent, the activating reagent includes N- hydroxyls Malaysia acyl Imines, carbodiimide and/or azodicarboxylate's activating reagent such as DCC, DIC, EDC, DEAD, DIAD etc..

In a preferred embodiment, comprising four at N by C₁-C₃Alkyl-substituted Hydroxypyridinone part and At least one coupling agent (such as those specifically described herein can be used for the octadentate chelating agent of the coupling moiety of hydroxy-acid group in end Any of) and activating reagent such as n-hydroxysuccinimide (NHS) activate, thus to form the NHS of octadentate chelating agent Ester.Activation (such as NHS) ester is separable or is used to being coupled to without separation and any has free amino (such as in lysine On side chain) tissue targeting part.Other Acibenzolars are well known in the art, and can be for effective leaving group (for example Fluorinated groups, tosylate group, methanesulfonic acid ester group, iodide etc.) any ester.But it is preferred that NHS esters.

Coupling reaction is carried out preferably within the shorter time and under about environment.1st step or the 2nd step coupling reaction it is normal Time is about 1 to 240 minute, preferably 5 to 120 minutes, more preferably 10 to 60 minutes.The conventional temperature of coupling reaction is 0 to 90 DEG C, more preferably preferably 15 to 50 DEG C, 20 to 40 DEG C.About 25 DEG C or about 38 DEG C are suitable.

Octadentate chelating agent is coupled into targeting moiety generally (or at least will not be irreversibly) can not adversely influence target Carried out under conditions of to the binding ability of part.Because bonding agent is typically based on the part of peptide or protein matter, this needs to compare Gentle condition with avoid denaturation or two grades/tertiary structure lose.It is preferred that aqueous conditions are (as discussed in all contexts ), and it is desirable that avoid extreme pH and/or redox.Therefore, step b) can be 3 to 10, preferably 4 to 9 in pH, More preferably 4.5 to 8 times progress.The neutrallty condition for redox can be needed, or reduces to avoid as mild as a dove Aoxidized in air.

Chelating agent available for the preferred tissue targeting of all aspects of the invention is AGC0018 specifically described herein. AGC0018 with²²⁷The complex compound of Th ions formed the present invention complex compound and its correspondingly preparation, purposes, method etc. it is preferred Embodiment.Other preferred embodiments used include being conjugated to tissue targeting in all such aspects of the present invention Partly AGC0019 (as described herein)²²⁷Th complex compounds, the part of tissue targeting include to CD22 acceptors, Any of FGFR2, mesothelin, HER-2, PSMA or CD33 have the monoclonal antibody of binding affinity.

Brief Description Of Drawings：

Fig. 1：Prove data of the EDTA/PABA to on-radiation antibody conjugates AGC1118 stabilization in solution.

Fig. 2：Shadow of the different buffers comprising the antibody HOPO conjugates with 10kGy radiation exposures to hydrogen peroxide level Ring.

Fig. 3：With up to about 8000Bq/ μ g specific activity²²⁷Th-AGC1118 (IRF measure) radioactive steady Change is acted on.

Fig. 4：With different total activities²²⁷Cytotoxicity (ginsengs of the Th-AGC1118 to Ramos (incubation times of 4 hours) See embodiment 3)

Fig. 5：²²⁷Th-AGC0718 induces the target specific cells of CD33 positive cells to kill (referring to embodiment in vitro 4)

Fig. 6：²²⁷Cytotoxicities of the Th-AGC0118 under high (20kBq/ μ g) and low (7.4kBq/ μ g) specific activity.It is negative Compare be with same dose scope, identical incubation time and read before number of days low combination peptide-albumin complex compound (referring to reality Apply example 5).

Fig. 7：²²⁷Th-AGC2518 induces the target specific cells of FGFR2 positive cells to kill (referring to implementation in vitro Example 6).

Fig. 8：²²⁷Th-AGC2418 induces the target specific cells of mesothelin positive cell to kill (referring to implementation in vitro Example 7).

Fig. 9：²²⁷Th-AGC1018 induces the targeting specific and dose dependent of PSMA positive LNCaP cells thin in vitro Born of the same parents are killed (referring to embodiment 9).

Now, the present invention is illustrated by following non-limiting example.All compounds of example are formed in embodiment The present invention preferred embodiment (including preferred intermediate and precursor) and can context allow any aspect in Be used alone or in any combination.Thus, for example, the compound 2 to 4 of embodiment 2, the compound 10 of embodiment 3 and reality Apply each in the compound 7 of example 4 or be completely formed its different types of preferred embodiment.

Embodiment 1

Synthesize the compound of formula (VIII)

Embodiment 1a)

Synthesize 2- (4- nitrobenzyls) dimethyl malenate

At 0 DEG C, sodium hydride (60% dispersant, 11.55g, 289mmol) is suspended in 450ml tetrahydrofurans (THF) In.Dimethyl malenate (40.0mL, 350mmol) was added dropwise in about 30 minutes.Reactant mixture is stirred 30 at 0 DEG C Minute.The 4- nitrobenzyls bromide (50.0g, 231mmol) being dissolved in 150mL THF is added dropwise at 0 DEG C in about 30 minutes, Then it is added dropwise at ambient temperature in two hours.

Add 500mL ethyl acetate (EtOAc) and 250mL NH₄Cl (aq, sat), then filters the solution.Separation is each Phase.With 2*250mL EtOAc aqueous phase extracteds.Organic phase is merged, with 250mL salt water washings, Na is used₂SO₄Dry, filter and Decompression is lower to remove solvent.

300mL heptane and 300mL methyl tertiary butyl ether(MTBE)s (MTBE) are added into residue and added to 60 DEG C.Filtering should Solution.Filtrate is placed in refrigerating chamber overnight and filtered.By filter cake is with 200mL heptane wash and is dried under reduced pressure, canescence is obtained Solid-like title compound.

Yield：42.03g, 157.3mmol, 68%.

1H-NMR(400MHz,CDCl3):3.30(d,2H,7.8Hz),3.68(t,1H,7.8Hz),3.70(s,6H), 7.36(d,2H,8.7Hz),8.13(d,2H,8.7Hz)。

Embodiment 1b)

Synthesize 2- (4- nitrobenzyls) propyl- 1,3- glycol

2- (4- nitrobenzyls) dimethyl malenates (28.0g, 104.8mmol) are dissolved in 560mL THF at 0 DEG C. Diisobutyl aluminium hydride (DIBAL-H) (1M in hexane, 420mL, 420mmol) is added dropwise in about 30 minutes at 0 DEG C.Will Reactant mixture is stirred two hours at 0 DEG C.

20mL water is added dropwise into reactant mixture at 0 DEG C.20mL NaOH are added dropwise into reactant mixture at 0 DEG C (aq, 15%), is then added dropwise 20mL water into reactant mixture.Mixture is stirred 20 minutes at 0 DEG C, then added about 150g MgSO₄.Mixture is stirred at room temperature 30 minutes, then filtered in Buchner funnel.By filter cake 500mL EtOAc is washed.By filter cake remove and with 800mL EtOAc and 200mL MeOH together stir about 30 minutes, then filtering solution. Merging filtrate is simultaneously dried under reduced pressure.

Gradients of the EtOAc in heptane is used on silica, then the DFC of the gradient using MeOH in EtOAc Obtain pale-yellow solid title compound.

Yield：15.38g, 72.8mmol, 69%.

1H-NMR(400MHz,CDCl3):1.97-2.13(m,3H),2.79(d,2H,7.6Hz),3.60-3.73(m, 2H),3.76-3.83(m,2H),7.36(d,2H,8.4Hz),8.14(d,2H,8.4Hz)。

Embodiment 1c)

Synthesize 2- (4- nitrobenzyls) propane -1,3- diyl bis-mesylates

2- (4- nitrobenzyls) propyl- 1,3- glycol (15.3g, 72.4mmol) is dissolved in 150mL CH at 0 DEG C₂Cl₂In. Triethylamine (23mL, 165mmol) is added, mesyl chloride (12mL, 155mmol) was then added dropwise in about 15 minutes, then Stir one hour at ambient temperature.

Add 500mL CH₂Cl₂, and use 2*250mL NaHCO₃(aq, sat), 125mL HCl (aq, 0.1M) and 250mL Salt water washing mixture.By organic phase Na₂SO₄Dry, filter and be dried under reduced pressure, obtain orange solids shape title compound.

Yield：25.80g, 70.2mmol, 97%.

1H-NMR(400MHz,CDCl3):2.44-2.58(m,1H),2.87(d,2H,7.7Hz),3.03(s,6H),4.17 (dd,2H,10.3,6.0Hz),4.26(dd,2H,10.3,4.4Hz),7.38(d,2H,8.6Hz),8.19(d,2H,8.6Hz)。

Embodiment 1d)

Synthesize (azane diyl two (ethane -2,1- diyls)) diamino acid di tert butyl carbonate

Imidazoles (78.3g, 1.15mol) is suspended in 500mL CH at room temperature₂Cl₂In.It is added portionwise into two dimethyl dicarbonate fourths Ester (Boc₂O)(262.0g,1.2mol).Reactant mixture is stirred at room temperature 1 hour.By reactant mixture 3*750mL Water washing, uses Na₂SO₄Dry, filter and volatile matter is removed under reduced pressure.

Dissolve the residue in 250ml toluene, and add diethylenetriamines (59.5mL, 550mmol).By reaction mixing Thing is stirred two hours at 60 DEG C.

Add 1L CH₂Cl₂, and with 2*250mL water washing organic phases.By organic phase Na₂SO₄Dry, filter and subtracting Pressure decrement.On silica using methanol (MeOH) in CH₂Cl₂Colorless solid is obtained with the DFC of the gradient in triethylamine Title compound.

Yield：102g, 336mmol, 61%.

¹H-NMR(400MHz,CDCl3):1.41(s,18H),1.58(bs,1H),2.66-2.77(m,4H),3.13- 3.26(m,4H),4.96(bs,2H)。

Embodiment 1e)

Synthesize (((2- (4- nitrobenzyls) propane -1,3- diyls) two (bases of azane three)) four (ethane -2,1- diyls)) four The tert-butyl ester of carbamic acid four

By 2- (4- nitrobenzyls) propane -1,3- diyls bis-mesylate (26.0g, 71mmol) and the ((second of azane diyl two Alkane -2,1- diyls)) diamino acid di tert butyl carbonate (76.0g, 250mmol) is dissolved in 700mL acetonitriles.Add N, N- diisopropyls Base ethamine (43mL, 250mmol).Reactant mixture is stirred under reflux 4 days.

Volatile matter is removed under reduced pressure.

The DFC of the gradient using EtOAc in heptane obtains light yellow solid foam-like title compound on silica Thing.

Yield：27.2g, 34.8mmol, 49%.

¹H-NMR(400MHz,CDCl3):1.40(s,36H),1.91-2.17(m,3H),2.27-2.54(m,10H), 2.61-2.89(m,2H),2.98-3.26(m,8H),5.26(bs,4H),7.34(d,2H,8.5Hz),8.11(d,2H, 8.5Hz)。

Embodiment 1f)

Synthesize N¹,N¹'-(2- (4- nitrobenzyls) propane -1,3- diyls) two (N¹- (2- amino-ethyls) second -1,2- two Amine), AGC0020

By (((2- (4- nitrobenzyls) propane -1,3- diyls) two (bases of azane three)) four (ethane -2,1- diyls)) four ammonia The tert-butyl ester of base formic acid four (29.0g, 37.1mmol) is dissolved in 950mL MeOH and 50mL water.At 30 DEG C in about 20 minutes dropwise Add chloroacetic chloride (50mL, 0.7mol).Reactant mixture is stirred overnight.

Volatile matter is removed under reduced pressure and dissolves the residue in 250mL water.Add 500mL CH₂Cl₂, then add 175mL NaOH (aq, 5M, with NaCl saturations).Each phase is separated, and uses 4*250mL CH₂Cl₂Aqueous phase extracted.Organic phase is merged, used Na₂SO₄Dry, filter and be dried under reduced pressure, obtain sticky rufous title compound as oil.

Yield：11.20g, 29.3mmol, 79%.Purity (HPLC Fig. 9)：99.3%.

¹H-NMR(300MHz,CDCl₃):1.55(bs,8H),2.03(dt,1H,6.6,13.3Hz),2.15(dd,2H, 12.7,6.6),2.34-2.47(m,10H),2.64-2.77(m,10H),7.32(d,2H,8.7Hz),8.10(d,2H, 8.7Hz).

¹³C-NMR(75MHz,CDCl₃):37.9,38.5,39.9,58.0,58.7,123.7,130.0,146.5,149.5

Embodiment 1g)

Synthesize 5- hydroxyl -6- oxo -1,2,3,6- tetrahydropyridine -4- carboxylic acid, ethyl esters

2-Pyrrolidone (76mL, 1mol) and diethy-aceto oxalate (140mL, 1.03mol) are dissolved in 1L toluene at room temperature In.Potassium ethoxide (EtOK) (24% in EtOH, 415mL, 1.06mol) is added, and reactant mixture is heated to 90 DEG C.

Due to reactant mixture retrogradation, 200mL EtOH are added portionwise into during first hour of reaction.Reaction is mixed Compound is stirred overnight and is cooled to room temperature.While stirring, 210mL HCl (5M, aq) are slowly added into.

200mL salt solution and 200mL toluene are added, and is separated each.With 2 × 400mL CHCl₃Aqueous phase extracted.It will merge Organic phase dry (Na₂SO₄), filter and reduce in a vacuum.Residue is recrystallized from EtOAc, light yellow solid is obtained Shape title compound.

Yield：132.7g, 0.72mol, 72%.

Embodiment 1h)

Synthesize 3- hydroxyl -2- oxo -1,2- dihydroxy-pyridine -4- carboxylic acid, ethyl esters

{ 5- hydroxyl -6- oxo -1,2,3,6- tetrahydropyridine -4- carboxylic acid, ethyl esters } (23.00g, 124.2mmol) is dissolved in In 150mL paraxylene, and add the palladium (10%, 5.75g) of carbon load.Reactant mixture is stirred overnight under reflux.It is cold But to after room temperature, reactant mixture is diluted and passed through with 300mL MeOHShort pad filtering.Washed with 300mL MeOH Wash the pad.Solvent is removed in vacuum, light reddish brown color solid-like title compound is obtained.

Yield：19.63g, 107.1mmol, 86%.MS(ESI,pos):206.1[M+Na]⁺,389.1[2M+Na]⁺

Embodiment 1i)

Synthesize 3- methoxyl group -1- methyl -2- oxo -1,2- dihydropyridine -4- carboxylic acid, ethyl esters

{ 3- hydroxyl -2- oxo -1,2- dihydropyridine -4- carboxylic acid, ethyl esters } (119.2g, 0.65mol) is dissolved at room temperature In 600mL dimethyl sulfoxides (DMSO) and 1.8L acetone.Add K₂CO₃(179.7g,1.3mol).At room temperature in about 1 hour dropwise Add the iodomethane (MeI) (162mL, 321mmol) being dissolved in 600mL acetone.

Reactant mixture is stirred at room temperature two hours, then adds MeI (162mL, 2.6mol).By reaction mixing Thing is stirred overnight under reflux.Reactant mixture is reduced under reduced pressure and 2.5L EtOAc are added.

Mixture is filtered and reduced under reduced pressure.By in SiO₂The dry post of gradients of the upper use EtOAc in heptane Flash chromatography (DFC) is purified, and obtains title compound.

Yield：56.1g, 210.1mmol, 32%.MS(ESI,pos):234.1[M+Na]⁺,445.1[2M+Na]⁺

Embodiment 1j)

Synthesize 3- (benzyloxy) -1- methyl -2- oxo -1,2- dihydropyridine -4- carboxylic acid, ethyl esters

At -78 DEG C, by { 3- methoxyl group -1- methyl -2- oxo -1,2- dihydropyridine -4- carboxylic acid, ethyl esters } (5.93g, 28.1mmol) it is dissolved in 80mL dichloromethane (DCM), and the BBr being dissolved in 20mL DCM is added dropwise₃(5.3mL, 56.2mmol).Reactant mixture is stirred 1 hour at -78 DEG C, the reaction is then heated to 0 DEG C.By the way that uncle 25mL is added dropwise Butyl methyl ether (tert-BuOMe) and 25mL MeOH terminating reactions.Volatile matter is removed in vacuum.Dissolve the residue in 90mL DCM With in 10mLMeOH and pass through SiO₂Short pad filtering.The pad is washed with solution of the 200mL 10%MeOH in DCM.In vacuum Lower removing volatile matter.Dissolve the residue in 400mL acetone.Add K₂CO₃(11.65g,84.3mmol)、KI(1.39g, 8.4mmol) with benzyl bromide a-bromotoluene (BnBr) (9.2mL, 84.3mmol).Reactant mixture is stirred overnight under reflux.Reaction is mixed Compound is diluted with 200mL EtOAc and uses 3x50mL water and 50mL salt water washings.The aqueous phase of merging is extracted with 2x50mL EtOAc Take.The organic phase of merging is dried into (Na₂SO₄), filter and remove volatile matter under vacuo, and in SiO₂In upper use heptane EtOAc (40-70%), by dry post purified by flash chromatography, obtains title compound as eluent.

Yield：5.21g, 18.1mmol, 65%.MS(ESI,pos):310.2[M+Na]⁺,597.4[2M+Na]⁺

Embodiment 1k)

Synthesize 3- (benzyloxy) -1- methyl -2- oxo -1,2- dihydropyridine -4- carboxylic acids

By { 3- (benzyloxy) -1- methyl -2- oxo -1,2- dihydropyridine -4- carboxylic acid, ethyl esters } (27.90g, 97.1mmol) It is dissolved in 250mL MeOH, and adds 60mL NaOH (5M, aq).Reactant mixture is stirred at room temperature 2 hours, then will Reactant mixture is concentrated under vacuum to about 1/3.Residue is diluted with 150mL water, and it is sour with hydrogen chloride (HCl) (5M, aq) It is 2 to change to pH.Sediment is filtered out and is dried in vacuo, colorless solid title compound is obtained.Yield：22.52g, 86.9mmol, 89%.

Embodiment 1l)

Synthesize 3- (benzyloxy) -1- methyl -4- (2- Thioxothiazolidin -3- carbonyls) pyridine -2 (1H) -one (AGC0021)

By { 3- (benzyloxy) -1- methyl -2- oxo -1,2- dihydropyridine -4- carboxylic acids } (3.84g, 14.8mmol), 4- bis- Dimethylaminopyridine (DMAP) (196mg, 1.6mmol) and 2- thiazoline -2- mercaptan (1.94g, 16.3mmol) are dissolved in 50mL In DCM.Add N, N'- dicyclohexylcarbodiimides (DCC) (3.36g, 16.3mmol).Reactant mixture is stirred overnight.Will Reaction filtering, solid is washed with DCM, and filtrate is reduced under vacuo.The yellow solid of gained is weighed from isopropanol/DCM Crystallization, obtains AGC0021.Yield：4.65g, 12.9mmol, 87%.MS(ESI,pos):383[M+Na]⁺,743[2M+Na]⁺

Embodiment 1m)

Synthesize AGC0023

By AGC0020 (8.98g；23.5mmol) it is dissolved in CH₂Cl₂In (600mL).Add AGC0021 (37.43g； 103.8mmol).Reaction is stirred at room temperature 20 hours.Reactant mixture is concentrated under reduced pressure.

In SiO₂Upper use methanol is in EtOAc and CH₂Cl₂1:The DFC of gradient in 1 mixture obtains solid foam AGC0023.

Average yield：26.95g, 20.0mmol, 85%.

Embodiment 1n)

Synthesize AGC0024

By AGC0023 (26.95g；20.0mmol) it is dissolved in ethanol (EtOH) (675mL).Add iron (20.76g； 0.37mol) and NH₄Cl(26.99g；0.50mol), water (67mL) is then added.Reactant mixture is stirred at 70 DEG C to two small When.Add iron (6.75g；121mmol), and by reactant mixture stirred one hour at 74 DEG C.Add iron (6.76g； 121mmol), and by reactant mixture stirred one hour at 74 DEG C.Reactant mixture is cooled down, then reactant mixture existed Decompression is lower to reduce.

In SiO₂Upper use methanol is in CH₂Cl₂In the DFC of gradient obtain solid foam AGC0024.

Yield：18.64g, 14.2mmol, 71%.

Embodiment 1o)

Synthesize AGC0025

By AGC0024 (18.64g；14.2mmol) it is dissolved in CH₂Cl₂In (750mL) and it is cooled to 0 DEG C.Add BBr₃(50g； 0.20mol), and by reactant mixture stir 75 minutes.While stirring for 0 DEG C, by carefully adding methanol (MeOH) (130mL) terminating reaction.Volatile matter is removed under reduced pressure.HCl (1.25M in EtOH, 320mL) is added into residue.Then, exist Flask is rotated 15 minutes using Rotary Evaporators under atmospheric pressure and environment temperature, volatile matter is then removed under reduced pressure.

The C closed in non-end₁₈Obtained on silica using the DFC of acetonitrile (ACN) Yu Shuizhong gradient to be slightly orange The AGC0025 of the vitreous solid of color.

Yield 13.27g, 13.9mmol, 98%.

Embodiment 1p)

Synthesize AGC0019

At room temperature by AGC0025 (10.63g；11.1mmol) it is dissolved in ACN (204mL) and water (61mL).Add amber Acid anhydrides (2.17g；21.7mmol), and by reactant mixture stir two hours.Reactant mixture is depressurized and reduced.In non-end envelope The C closed₁₈On silica jade-green vitreous solid is obtained using the DFC of gradients of the ACN in water.

The solid is dissolved in MeOH (62mL) and water (10.6mL) at 40 DEG C.Lower the solution is added dropwise ultrasonically treated Into EtOAc (750mL).Sediment is filtered, is washed and is dried under reduced pressure with EtOAc, is obtained with the greyish white of light green color tone Color solid-like AGC0019.

Yield：9.20g, 8.7mmol, 78%.H-NMR(400MHz,DMSO-d₆),¹³C-NMR(100MHz,DMSO-d₆)。

Embodiment 2

The pure thorium -227 of separation

Thorium -227 is separated from the generator of actinium -227.Actinium -227 irradiates radium-226, subsequent radium -227 by thermal neutron (t1/2=42.2m) decay to actinium -227 and produce.In 8M HNO₃Declined in solution by anion-exchange chromatography by actinium -227 Become mixture selective retention thorium -227.The use of internal diameter is the post that 2mm, length are 30mm, it contains 70mg1-X8 resins (200-400 mesh, nitrate salts).Elute after actinium -227, radium -223 and daughter, extracted with 12M HCl from post from post Go out thorium -227.The eluent of thoriated -227 is evaporated to drying, and residue is resuspended in 0.01M before markers step In HCl.

Embodiment 3

²²⁷Cytotoxicities of the Th-AGC1118 to Ramos

Embodiment 3a)

Produce anti-CD22 monoclonal antibody (AGC1100)

Monoclonal antibody (mAb) hLL2's --- being also referred to as epratuzumab (being expressed as AGC1100 herein) --- Sequence, can be such as Leung, Goldenberg, Dion, Pellegrini, Shevitz, Shih, and Hansen:Molecular Immunology 32:Structure described in 1413-27,1995.

In embodiments of the invention mAb used is by Immunomedics Inc, prepared by New Jersey, USA.Should MAb can be for example in Chinese hamster ovary suspension (CHO-S) cell with the plasmid transfection comprising coding light chain and the gene of heavy chain It is middle to prepare.The first stable clone is selected to be used to use standardization program., can in disposable bioreactor after about 14 days Monoclonal antibody is collected after filtering supernatant.AGC1100 can by a-protein affinity chromatography (MabSelect SuRe, Atoll, Weingarten/Germany), ion-exchange step is then carried out to be further purified.Using based on electrostatic and hydrophobic Property the 3rd purification step remove aggregation and may remaining impurity.Pass through isoelectric focusing, SDS-PAGE analyses, N- ends End sequencing and LC/MS analyze to determine AGC1100 homogeneity.Sample purity further passes through SEC (SEC) To analyze.

Embodiment 3b)

MAb AGC1100 (epratuzumab) and chelating agent AGC0019 (compound of formula (VIII)) is coupled to obtain Conjugate AGC1118

Before conjugated, pH 7.5 PB is added to antibody-solutions (AGC1100) to improve the slow of solution Rush ability.Determine AGC1100 in container (mAb) amount.

Chelating agent AGC0019 is dissolved in 1:1 DMA:In 0.1M MES buffer solutions (pH is 5.4).NHS and EDC are dissolved in PH is in 5.4 0.1M MES buffer solution.

Prepare chelating agent/n-hydroxysuccinimide (NHS)/1- ethyls -3- (3- dimethylaminopropyls) carbodiimide (EDC) 1/1/3 molar equivalent solution is to activate the chelating agent.In order to antibody conjugate, by mol ratio be 7.5/7.5/22.5/ The chelating agent of the activation of 1 (chelating agent/NHS/EDC/mAb) is added in mAb.After 20-40 minutes, with 12%v/v 0.3M lemons Acid terminates conjugation reaction, so as to which pH is adjusted into 5.5.

Then solution is buffered by tangential flow filtration (Tangential Flow Filtration) with constant volume and handed over 30mM citrates, 70mM NaCl, 2mM EDTA, 0.5mg/ml pABA, pH are shifted to in 5.5 (TFF buffers).In diafiltration At the end of, solution is poured into preparation vessels.With TFF buffers (30mM citrates, 70mM NaCl, 2mM EDTA, 0.5mg/ml pABA, pH 5.5) and 7%w/v polysorbate80s prepare product to obtain 2.6mg/mL in 30mM citric acids AGC1118 in salt, 70mM NaCl, 2mM EDTA, 0.5mg/mL pABA, 0.1%w/v PS80, pH 5.5.Finally, will be molten Liquid is filtered into aseptic bottle by 0.2 μm of filter, is then stored.

Embodiment 3c)

Prepare²²⁷The dosage of Th-AGC1118 injections

The one bottle 20MBq chloride film of thorium -227 is dissolved in 2ml 8M HNO₃In solution, place 15 minutes, Ran Houqu Go out solution, applied on anion-exchange column, to remove the increased radium -223 with the time.With 3ml 8M HNO₃With 1ml washings The post is washed, then with 3ml 3M HCl elution thoriums -227.The elution activity of thorium -227 is measured, 10MBq dosage is transferred to sky In 10ml vials.Then acid is evaporated using vavuum pump, and bottle is placed in heating zone (being set as 120 DEG C) 30-60 minutes. Reach after room temperature, adding 6ml 2.5mg/ml AGC1118 conjugates is used for radioactive label.Bottle is slowly mixed, room Temperature is lower to place 15 minutes.Then solution is sterile filtered into sterile vials, using preceding sampling, carries out iTLC analyses to determine RCP。

Embodiment 3d)

With different total activities and specific activity²²⁷Cytotoxicities of the Th-AGC1118 to Ramos

In our current research, tested by changing total activity and specific activity under 4 hours incubation times²²⁷Th-AGC1118 Dosage.This research is in 96 AND DEWATERING FOR ORIFICE STRUCTUREs with 10/50kBq/ μ g specific activity and 5,10,20 and 40kBq/ml total activity Carry out.

Ramos cells are cultivated in the RPMI1640- culture mediums with 10%FBS and 1% penicillin/streptomycin (the 22 sections).Cell is transferred in centrifuge tube and centrifuged 5 minutes under 300G, and is suspended in 5mL culture mediums, then in Z2 storehouses Counted on your special counter (Z2Coulter Counter).Cell suspending liquid is diluted into cell concentration with culture medium is In 400.000 cell/ml, and 48 holes (200 μ l/ holes) being transferred in 96 orifice plates (80.000 cells/wells).Use CellTiter-Glo luminescent cells vitality test (Promega) measures cell viability.Referring to Fig. 4.

Embodiment 4

²²⁷Cytotoxicities of the Th-AGC0718 to HL-60

Embodiment 4a)

Produce anti-CD 33 monoclonal antibody (AGC0700)

The sequence of monoclonal antibody (mAb) HuM195/ lintuzumabs (being expressed as AGC0700 herein), can be from such as (1) (2) retrieve and obtain in the document described in.CobraBiologics (Sweden prepared in equipment) AGC0700.In short, using VectorSoftware (Invitrogen/Life-Technologies Ltd., Paisley, Britain) by the amino acid sequence reverse translation of heavy chain and light chain into DNA sequence dna.Remove C- ends from IgG1 heavy chain genes The codon of lysine (Lys) is held, to contribute to the accurate survey of conjugate and antibody ratio (CAR) listed by embodiment 2 It is fixed.By CobraBiologics (Sweden), the DNA sequence dna of gained is carried out into codon optimization is used for In mammalian cell express, and by GeneArt (GeneArt/Life-Technologies Ltd., Paisley, United Kingdom) synthesis, one-step cloning is gone forward side by side into expression vector.Chinese hamster ovary suspension (CHO-S) cell is made With coding AGC0700 V_H- and V_LThe plasmid stabilisation transfection of-domain, and supplemented with puromycin (12.5mg/l；Sigma Aldrich CD-CHO culture mediums (the Invitrogen/Life-Technologies Ltd., Paisley, United of standard) Kingdom grown in the presence of).Pass through the stable clone of limiting dilution assay screening expression AGC0700 in 25 generations.Clone stability leads to The protein titre of measurement supernatant is crossed to assess.Set up the cell bank of most stable clone and carry out Cord blood.

At 37 DEG C, mAb expression about 14 days is carried out in 250L disposable bioreactor.After filtering supernatant, collect Monoclonal antibody.AC0700 enters one through a-protein affinity column (MabSelect SuRe, Atoll, Weingarten/Germany) Step purifying, then using an anion-exchange chromatography (QFF-Sepharose；GE Healthcare) and a cation friendship (PorosXS is composed in colour changing；Invitrogen/Life-Technologies Ltd.) improve purity and ultimate yield.AGC0700 Homogeneity determined by isoelectric focusing and SDS-PAGE analyses.In fixed CD33-Fc targets (Novoprotein) With reference to the activity for the AGC0700 that purifying is analyzed in ELISA.Sample purity is analyzed by SEC (SEC).

Bibliography

(1)Scheinberg DA.“Therapeutic uses of the hypervariable region of monoclonal antibody M195and constructs thereof.US Patent Application 6007814 (1999Dec 28).

(2)Co MS et al；J Immunol.1992Feb 15；148(4):1149-54.Chimeric and humanized antibodies with specificity for the CD33antigen.

Embodiment 4b)

MAb AGC0700 (lintuzumab) and chelating agent AGC0019 (compound of formula (VIII)) is coupled to obtain Conjugate AGC0718

As described in Example 3, it is conjugated, but has a little exception.

Before conjugated, pH 7.5 PB is added to antibody-solutions (AGC0700) to improve the slow of solution Rush ability.Determine AGC0700 in container (mAb) amount.

Chelating agent AGC0019 is dissolved in 1:1 DMA:In 0.1M MES buffer solutions (pH is 5.4).NHS and EDC are dissolved in PH is in 5.4 0.1M MES buffer solution.

Chelating agent/NHS/EDC 1/1/3 molar equivalent solution is prepared to activate the chelating agent.In order to antibody conjugate, will Mol ratio is added in mAb for the chelating agent of the activation of 20/20/60/1 (chelating agent/NHS/EDC/mAb).After 40-60 minutes, Conjugation reaction is terminated with 12%v/v 0.3M citric acids, so as to which pH is adjusted into 5.5.

Then by solution by tangential flow filtration with constant volume buffering exchange to 30mM citrates, 154mM NaCl, 2mM EDTA, 2mg/ml pABA, pH is in 5.5 (TFF buffers).At the end of diafiltration, solution is poured into preparation vessels. Prepare product to obtain with TFF buffers (30mM citrates, 154mM NaCl, 2mM EDTA, 2mg/ml pABA, pH 5.5) To AGC0718s of the 2.5mg/mL in 30mM citrates, 154mM NaCl, 2mM EDTA, 2mg/mL pABA, pH 5.5.Most Afterwards, solution is filtered into aseptic bottle by 0.2 μm of filter, then stored.

Embodiment 4c)

Prepare²²⁷The dosage of Th-AGC0718 injections

The one bottle 20MBq chloride film of thorium -227 is dissolved in 2ml 8M HNO₃In solution, place 15 minutes, Ran Houqu Go out solution, applied on anion-exchange column, to remove the increased radium -223 with the time.With 3ml 8M HNO₃With 1ml washings The post is washed, then with 3ml 3M HCl elution thoriums -227.The elution activity of thorium -227 is measured, 10MBq dosage is transferred to sky In 10ml vials.Then acid is evaporated using vavuum pump, and bottle is placed in heating zone (being set as 120 DEG C) 30-60 minutes. Reach after room temperature, adding 6ml 2.5mg/ml AGC0718 conjugates is used for radioactive label.Bottle is slowly mixed, room Temperature is lower to place 15 minutes.Then solution is sterile filtered into sterile vials, using preceding sampling, carries out iTLC analyses to determine RCP。

Embodiment 4d)

With different total activities²²⁷Cytotoxicities of the Th-AGC0718 to HL-60

In order to prove after CD33+ cells are combined²²⁷Th-AGC0718 cytotoxicity, carries out vitro cytotoxicity measure. For the purpose of it, human marrow knurl leukemia HL-60 cell line and CD33 negative B cells system (Ramos) are exposed to²²⁷In Th-AGC0718.2 and 20kBq/ml total activity is determined under 44kBq/ μ g specific activitys.All experimental methods are recorded in In RD2013.093.In short, preparing 50 000 people in IMDM culture mediums with 10%FBS and 1% penicillin/streptomycin HL-60 cells/ml, and be seeded in the density of 100.000 cells/wells in 24 orifice plates.At 37 DEG C, lived with 0-20kBq/ml Degree²²⁷Th-AGC0718 incubated cells 4 hours.It is parallel to prepare each²²⁷Th- Isotype control conjugate samples and ab are unlabelled AGC0718 samples, are used as respective control.Then cell is washed with fresh culture, and is seeded to 24 new well culture plates In.

On different time points, cell is collected, vigor is determined using CellTiterGlo kits (Promega).It is living Power is represented by the way that positive control (untreated cell) is set as into 100% with %.Referring to Fig. 5.

Embodiment 5

²²⁷Cytotoxicities of the Th-AGC0118 to SKOV-3

Embodiment 5a)

Synthesize AGC0100 (Herceptin)

Trastuzumab monoclonal antibody (being expressed as AGC0100 herein) is bought from Roche, and is dissolved in PBS (Dulbecco BIOCHROM in), concentration is 10mg/ml.

Embodiment 5b)

MAb AGC0100 (Herceptin) and chelating agent AGC0019 (compound of formula (VIII)) is coupled to obtain Conjugate AGC0118

As described in Example 3, it is conjugated, there is a small amount of modification.The TFF purifying solvent resistant columns for the mAb being finally conjugated Chromatogram is replaced.

Phosphate buffers of the pH for 7.4 11%1M is added in Herceptin into PBS.By chelating agent (AGC0019) NHS and EDC are dissolved in and embodiment 3b) described in same solution in.During activation, chelating agent/NHS/EDC's Mol ratio is 1/1/3.Corresponding chelating agent/NHS/EDC/mAb 8/8/25/1 mol ratio and 30-40 minutes it is conjugated when Between produce conjugated AGC0118 0.7-0.9 CAR (chelating agent and antibody ratios).By the lemon for adding 12%v/v 0.3M Lemon acid terminating reaction so that final pH is 5.5.

By withSuperdex 200 (GE Healthcare) post of system (GE Healthcare) connection Upper gel filtration, carry out AGC0118 conjugates purifying and and buffer exchange to 30mM citrates (pH 5.5), 154mM NaCl.Protein concentration is measured under Abs 280nm, then product is prepared (to obtain 2.5mg/mL's with buffer AGC0118, pH 5.5 in 30mM citrates, 154mM NaCl, 2mM EDTA, 2mg/mL pABA).Finally, by solution It is filled into aseptic bottle, is then stored by 0.2 μm of filter.

Embodiment 5c)

Prepare²²⁷The dosage of Th-AGC0118 injections

It is marked as previously described：

The one bottle 20MBq chloride film of thorium -227 is dissolved in 2ml 8M HNO3 solution, placed 15 minutes, Ran Houqu Go out solution, applied on anion-exchange column, to remove the increased radium -223 with the time.With 3ml 8M HNO₃With 1ml washings The post is washed, then with 3ml 3M HCl elution thoriums -227.The elution activity of thorium -227 is measured, 10MBq dosage is transferred to sky In 10ml vials.Then acid is evaporated using vavuum pump, and bottle is placed in heating zone (being set as 120 DEG C) 30-60 minutes. Reach after room temperature, adding 6ml 2.5mg/ml AGC0118 conjugates is used for radioactive label.Bottle is slowly mixed, room Temperature is lower to place 15 minutes.Then solution is sterile filtered into sterile vials, using preceding sampling, carries out iTLC analyses to determine RCP。

Embodiment 5d)

With different total activities²²⁷Cytotoxicities of the Th-AGC0118 to SKOV-3

It is added to the total activity in hole by changing during 4 hours incubation times and tests various dosage²²⁷Th- AGC0118 cytotoxicity.In experiment the previous day, SKOV-3 cells are inoculated in 96 orifice plates with 10000/hole.The 1st My god, it is 5,10,20 and 40kBq/ml chelating by a series of total activities in the case where specific activity is 20kBq/ μ g²²⁷Th-AGC0118 It is added in cell.After incubation period terminates, remaining be not associated with is removed by many array pipettes²²⁷Th-AGC0118, Ran Houyong Culture medium is once extraly washed, then is washed with fresh culture.With 10%FBS and 1% penicillin/strepto- SKOV-3 cells are cultivated in the Mc-Coy culture mediums of element.With²²⁷Th-AGC0118 incubation periods, are substituted with serum free medium The culture medium.At the 4th day, cell viability is measured with CellTiter-Glo luminescent cells vitality test (Promega).Ginseng See Fig. 6.

Embodiment 6

²²⁷Cytotoxicities of the Th-AGC0118 to NCI-H716

Embodiment 6a)

Produce FGFR2 monoclonal antibodies (BAY1179470；AGC2500)

Monoclonal antibody BAY 1179470 (being also shown as AGC2500 herein) generation is recorded in detail In WO2013076186A1.In short, obtaining antibody by biopanning (biopanning) on FGFR2 antigens.Gained The antibody of IgG 1 is expressed in Chinese hamster ovary celI, and using a-protein affinity column (MAb Select Sure) purifying, is then passed through SEC carrys out separating monomer fraction.Antibody is formulated in the PBS that pH is 7.4.Analytic type SEC proves uniformity> 99%.

Embodiment 6b)

MAb AGC2500 and chelating agent AGC0019 (formula (VIII) compound) are coupled to obtain conjugate AGC2518

It is 7.5 that solution containing antibody, which is adjusted to pH,.Chelating agent AGC0019 is dissolved in 1:1 DMA:PH is 5.4 0.1M In MES buffer solutions.NHS and EDC are dissolved in the 0.1M MES buffer solutions that pH is 5.4.Prepare the 1/1/3 of chelating agent/NHS/EDC Molar equivalent solution is to activate the chelating agent.In order to antibody conjugate, by mol ratio be 10/10/30/1 (chelating agent/NHS/ EDC/mAb the chelating agent of activation) is added in mAb.After 30 minutes, conjugation reaction is terminated with 12%v/v0.3M citric acids, with Just pH is adjusted to 5.5., as mobile phase, response sample is then added to for 5.5 with 30mM citrates, 70mM NaCl, pH On HiLoad 16/600Superdex 200 (prep-grade) post, with separating monomer fraction., will at the end of chromatographic isolation Antibody conjugates AGC2518 concentrations are in 30mM citrates, 70mM NaCl, 2mM EDTA and 0.5mg/ml pABA 2.5mg/ml.All operations are recorded in RD.2014.092, Journal No.211/149,140619AEF.

Embodiment 6c)

Prepare²²⁷The dosage of Th-AGC2518 injections

The one bottle 20MBq chloride film of thorium -227 is dissolved in 2ml 8M HNO3 solution, placed 15 minutes, Ran Houqu Go out solution, applied on anion-exchange column, to remove the increased radium -223 with the time.With 3ml 8M HNO₃With 1ml washings The post is washed, then with 3ml 3M HCl elution thoriums -227.The elution activity of thorium -227 is measured, 10MBq dosage is transferred to sky In 10ml vials.Then acid is evaporated using vavuum pump, and bottle is placed in heating zone (being set as 120 DEG C) 30-60 minutes. Reach after room temperature, adding 6ml 2.5mg/ml AGC2518 conjugates is used for radioactive label.Bottle is slowly mixed, room Temperature is lower to place 15 minutes.Then solution is sterile filtered into sterile vials, using preceding sampling, carries out iTLC analyses to determine RCP。

Embodiment 6d)

With different total activities²²⁷Cytotoxicities of the Th-AGC2518 to NCI-H716 cells

In order to prove after FGFR2+ cells are combined²²⁷Th-AGC2518 cytotoxicity, carries out vitro cytotoxicity survey It is fixed.For the purpose of it, human colorectal cancer cell system NCI-H716 is exposed to²²⁷In Th-AGC2518.In 2kBq/ μ g 2,10,20 and 40kBq/ml total activity is tested under specific activity.It is abreast similar to prepare incoherent Isotype control.All experiments Method is recorded in RD2014.138.In short, 400000 people NCI-H716 cells/ml in the culture mediums of RPMI 1640 Prepared, and be inoculated with the density of 80.000 cells/wells in 96 orifice plates with 10%FBS and 1% penicillin/streptomycin.37 At DEG C, with 0-40kBq/ml activity²²⁷Th-AGC2518 and respective²²⁷Th- Isotype control conjugate sample incubations cell 30 Minute.Then cell is washed with fresh culture, and be seeded in 96 new well culture plates.After 5 days and after 7 days, collect thin Born of the same parents, and use CellTiterGlo kits (Promega) measurement vigor.Vigor is by by positive control (untreated cell) Vigor is set as 100% and represented with %.Referring to Fig. 7.

Embodiment 7

²²⁷Cytotoxicities of the Th-AGC2418 to HT29 cells

Embodiment 7a)

Produce mesothelin monoclonal antibody (BAY 86-1903；AGC2400)

Monoclonal antibody BAY 86-1903 (also referred to herein as AGC2400) generation is recorded in detail In WO2009068204.In short, obtaining antibody by biopanning on mesothelin antigen.The antibody of IgG 1 of gained exists Express, purified using a-protein affinity column (MAb Select Sure), then using HIC posts (Toyopearl in Chinese hamster ovary celI Butyl 600M) remove aggregation.Antibody is formulated in the PBS that pH is 7.5.

Embodiment 7b)

MAb AGC2400 and chelating agent AGC0019 (compound of formula (VIII)) are coupled to obtain conjugate AGC2418

It is 7.5 that solution containing antibody, which is adjusted to pH,.Chelating agent AGC0019 is dissolved in 1:1 DMA:PH is 5.4 0.1M In MES buffer solutions.NHS and EDC are dissolved in the 0.1M MES buffer solutions that pH is 5.4.Prepare the 1/1/3 of chelating agent/NHS/EDC Molar equivalent solution is to activate the chelating agent.In order to antibody conjugate, by mol ratio be 16.5/16.5/49.5/1 (chelating agent/ NHS/EDC/mAb the chelating agent of activation) is added in mAb.After 30 minutes, terminated with 12%v/v 0.3M citric acids conjugated anti- Should, so as to which pH is adjusted into 5.5., as mobile phase, response sample is added again for 5.5 with 30mM citrates, 70mM NaCl, pH Onto HiLoad16/600Superdex 200 (prep-grade) post, with separating monomer fraction., will at the end of chromatographic isolation Antibody conjugates AGC2418 concentrations are in 30mM citrates, 70mM NaCl, 2mM EDTA and 0.5mg/ml pABA 2.5mg/ml.All operations are recorded in RD.2014.111, Journal No.211/160,140814AEF.

Embodiment 7c)

Prepare²²⁷The dosage of Th-AGC2418 injections

The one bottle 20MBq chloride film of thorium -227 is dissolved in 2ml 8M HNO3 solution, placed 15 minutes, Ran Houqu Go out solution, applied on anion-exchange column, to remove the increased radium -223 with the time.With 3ml 8M HNO₃With 1ml washings The post is washed, then with 3ml 3M HCl elution thoriums -227.The elution activity of thorium -227 is measured, 10MBq dosage is transferred to sky In 10ml vials.Then acid is evaporated using vavuum pump, and bottle is placed in heating zone (being set as 120 DEG C) 30-60 minutes. Reach after room temperature, adding 6ml 2.5mg/ml AGC2418 conjugates is used for radioactive label.Bottle is slowly mixed, room Temperature is lower to place 15 minutes.Then solution is sterile filtered into sterile vials, using preceding sampling, carries out iTLC analyses to determine RCP。

Embodiment 7d)

With different total activities²²⁷Cytotoxicities of the Th-AGC2418 to the HT29 cells of overexpression mesothelin antigen

In order to prove after mesothelin+cell is combined²²⁷Th-AGC2418 cytotoxicity, carries out vitro cytotoxicity survey It is fixed.For the purpose of it, the Human Colorectal Cancer cell line HT29 transfected with mesothelin antigen is exposed to²²⁷Th-AGC2418。 In the case where specific activity is 10kBq/ μ g, in the case where diluting three times, in 12 titrimetry total activities since 5kBq/ml. It is abreast similar to prepare incoherent Isotype control.All experimental implementations are recorded in RD2014.154.In short, in RPMI 200000 mankind HT29 cells/ml 10%FBS, 1% penicillin/chain of the use mesothelin antigen transfection in 1640 culture mediums Mycin, 1%NaHCO₃, 600 μ g/ml hygromycin Bs prepare, and be inoculated in the density of 40.000 cells/wells in 96 orifice plates. At 37 DEG C, with 0 to 40kBq/ml activity²²⁷Th-AGC2418 and respective²²⁷Th- Isotype control conjugate sample incubation cells 6 days.At the 6th day, cell is collected, and use CellTiterGlo kits (Promega) measurement vigor.Vigor is by by the positive Control (untreated cell) vigor is set as 100% and represented with %.

Embodiment 8

Compare the stability of acid amides and the conjugate of isothiocyanates-connection

At 40 DEG C, AGC1118 and the corresponding conjugate (AGC1115) with isothiocyanates coupling moiety are stored in 11 days in the aqueous solution.Periodically sampling.

It is normalized to 40 DEG C of samples of each 4 DEG C of sample points

It can be seen that the conjugate being coupled for acid amides does not observe measurable reduction of conjugate concentration from upper table.Phase Instead, isothiocyanates conjugate reduces by 12% after reducing by 8%, 11 days after 5 days.

Embodiment 9

Embodiment 9a)

Produce PSMA monoclonal antibodies (AGC1000)

PSMA monoclonal antibodies (hereinafter referred to AGC1000) are purchased from Progenics, USA.

Embodiment 9b)

MAb AGC1000 and chelating agent AGC0019 (compound of formula (VIII)) are coupled to obtain conjugate AGC1018

It is 7.5 that solution containing antibody, which is adjusted to pH,.Chelating agent AGC0019 is dissolved in 1:1 DMA:PH is 5.5 0.1M In MES buffer solutions.NHS and EDC are dissolved in the 0.1M MES buffer solutions that pH is 5.5.Prepare the 1/1/2 of chelating agent/NHS/EDC Molar equivalent solution is to activate the chelating agent.In order to antibody conjugate, by mol ratio be 20/20/40/1 (chelating agent/NHS/ EDC/mAb 10 minutes between 4 parts of the chelating agent of activation) point, every part, it is added in mAb.It is 7.3 with pH after 50 minutes 12%v/v 1M citric acids terminate conjugation reaction.Conjugate is purified and enters row buffering by tangential flow filtration (TFF) and is exchanged. The buffer solution of preparation is that 30mM citrates, 70mM NaCl, 2mM EDTA, 0.5mg/ml pABA, pH are 5.5.In diafiltration knot Shu Shi, solution is poured into (bulk) container in bulk, concentration is adjusted into 2.7mg/ml.Finally, bulk liquid solutions are passed through into 0.2 μm of nothing Bacterium filter is filtered, and is transferred in sterile vials for being stored at -20 DEG C.

Embodiment 9c)

Prepare²²⁷The dosage of Th-AGC1018 injections

The one bottle about 50MBq chloride film of thorium -227 is dissolved in 2ml 8M HNO₃In solution, placement 15 minutes, then Solution is taken out, applied on anion-exchange column, to remove the increased radium -223 with the time.With 3ml 8M HNO₃With 1ml water The post is washed, then with 3ml 3M HCl elution thoriums -227.HCl is eluted with the heating zone for being set to 100 DEG C using vavuum pump Liquid evaporates 60-90 minutes.The activity through dry Th-227 is measured in dose calibrator.Dry Th-227 is dissolved in In 0.05M HCl, 0.5MBq/ μ l concentration is obtained.To carry out radioactive label, conjugate is diluted in Formulation Buffer AGC1018 is to obtain the 25 μ g mAb in 200 μ l.1MBq Th-227 are mixed into AGC1018 solution, and in germanium detection Accurate Th-227 activity is measured on device.Allow chelating at room temperature 30-60 minutes, be then sterile filtered into sterile vials. Using preceding sampling, carry out iTLC analyses to determine RCP.

Embodiment 9d)

²²⁷Cytotoxicities of the Th-AGC1018 to expression PSMA LNCaP cells

In order to prove after PSMA positive cells are combined²²⁷Th-AGC1018 cytotoxicity, carries out vitro cytotoxicity survey It is fixed.For the purpose of it, human prostate cancer cell line LNCaP is exposed to²²⁷Th-AGC1018.It is 40kBq/ μ g in specific activity Under, in the case where diluting three times, in 12 titrimetry total activities since 20kBq/ml.Abreast prepare incoherent Isotype control.All experimental implementations are recorded in archive RD2015.101.In short, supplemented with 10%FBS and 1% mould People's LNCaP cells are cultivated in the culture mediums of RPMI 1640 of element/streptomysin.Cell is inoculated in the density of 2500 cells/wells In 96 orifice plates.24 hours after inoculation (the 1st day), at 37 DEG C, total activity is exposed cells to for 0 to 20kBq/ml's²²⁷Th- AGC1018 and²²⁷Th Isotype controls continue 5 days.At the 6th day, cell is collected, and use CellTiterGlo kits (Promega) vigor is measured.Vigor is represented by the way that positive control (untreated cell) vigor is set as into 100% with %.

Sequence table

<110>Bayer Co., Ltd

<120>Conjugation methods

<130> 95.121941

<160> 5

<170>PatentIn version 3s .5

<210> 1

<211> 113

<212> PRT

<213>Artificial sequence

<220>

<223>Mouse sequence of light chain

<400> 1

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Glu Asn Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ala Asn His Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Arg Val Gln Val Glu Asp Leu Ala Ile Tyr Tyr Cys His Gln

85 90 95

Tyr Leu Ser Ser Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 2

<211> 114

<212> PRT

<213>Artificial sequence

<220>

<223>Humanization sequence of light chain

<400> 2

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Val Asn Asp Arg Met Ser Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ala Asn His Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Lys Ala Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly

50 55 60

Val Pro Asp Ser Phe Thr Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu

65 70 75 80

Phe Ile Ser Arg Ser Leu Val Pro Asp Leu Ile Ile Thr Tyr Cys His

85 90 95

Gln Tyr Leu Ser Ser Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg

<210> 3

<211> 116

<212> PRT

<213>Artificial sequence

<220>

<223>Mouse sequence of heavy chain

<400> 3

Gln Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Ser Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Leu His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Arg Asn Asp Tyr Thr Glu Tyr Asn Gln Asn Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Ile Thr Thr Phe Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210> 4

<211> 116

<212> PRT

<213>Artificial sequence

<220>

<223>First humanization sequence of heavy chain

<400> 4

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Arg Asn Asp Tyr Thr Glu Tyr Asn Gln Asn Phe

50 55 60

Lys Asp Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Phe Cys

85 90 95

Ala Arg Arg Asp Ile Thr Thr Phe Tyr Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 5

<211> 116

<212> PRT

<213>Artificial sequence

<220>

<223>Second humanized heavy chain's sequence

<400> 5

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Arg Asn Asp Tyr Thr Glu Tyr Asn Gln Asn Phe

50 55 60

Lys Asp Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Phe Cys

85 90 95

Ala Arg Arg Asp Ile Thr Thr Phe Tyr Trp Gly Gln Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

Claims (27)

1. a kind of method for the thorium complex for forming tissue targeting, methods described includes：

A) formed and include four at N by C₁-C₃Alkyl-substituted Hydroxypyridinone (HOPO) is partly hydroxy-acid group with end The octadentate chelating agent of coupling moiety；

B) the octadentate chelating agent is coupled to by least one am amide coupling agent at least one comprising at least one amine moiety Tissue targeting peptide or protein matter on so that produce tissue targeting chelating agent；And

C) chelating agent by the tissue targeting is contacted with the aqueous solution of the ion of the thorium isotope comprising at least one transmitting α.

2. the method for claim 1 wherein step b) is carried out in aqueous.

3. the method for claim 1 or 2, wherein the am amide coupling agent is in aqueous to be functional.

4. the method for any one of preceding claims, wherein the am amide coupling agent is carbodiimide coupling agent, such as 1- second Base -3- (3- dimethylaminopropyls) carbodiimide (EDC), N, N '-DIC (DIC) or the hexamethylene of N, N '-two Base carbodiimide (DCC).

5. the method for any one of preceding claims, wherein step b) are carried out in pH is 4 to 9 aqueous solution.

6. the method for any one of preceding claims, wherein step b) are carried out 5 to 120 minutes at 15 to 50 DEG C.

7. the method for any one of preceding claims, wherein step c) are carried out 1 to 60 minute at 15 to 50 DEG C.

8. the method for any one of preceding claims, wherein the octadentate chelating agent includes four 3,2-HOPO parts.

9. the method for any one of preceding claims, wherein the octadentate chelating agent is selected from formula (VIb) and (VII)：

Wherein Rc is the junction portion that end is carboxylic moiety, for example

[-CH₂- Ph-N (H)-C (=O)-CH₂-CH₂- C (=O) OH],

[-CH₂-CH₂- N (H)-C (=O)-(CH₂-CH₂-O)_1-3-CH₂-CH₂- C (=O) OH] or

[-(CH₂)_1-3- Ph-N (H)-C (=O)-(CH₂)_1-5- C (=O) OH],

Wherein Ph is phenylene, preferred pair phenylene.

10. the method for any one of preceding claims, wherein the tissue target to part be monoclonal antibody or polyclonal Antibody, antibody fragment (such as Fab, F (ab ')₂, Fab ' or scFv), or this antibody-like and/or fragment construct.

11. the method for any one of preceding claims, wherein the tissue target to part to CD22 acceptors, FGFR2, mesothelium Element, HER-2, PSMA or CD33 have binding affinity.

12. the thorium complex for method formation or the tissue targeting that can be formed for passing through any one of claim 1 to 11.

13. the thorium complex of the tissue targeting of claim 12, it includes four 3,2-HOPO parts.

14. the thorium complex of the tissue targeting of claim 12 or claim 13, its to CD22 acceptors, FGFR2, mesothelin, HER-2, PSMA or CD33 have binding affinity.

15. the thorium complex of the tissue targeting of any one of claim 12 to 14, it includes transmitting α thorium radionuclide Such as²²⁷Th 4+ ions.

16. the thorium complex of the tissue targeting of any one of claim 12 to 15, its octadentate comprising formula (VIb) or (VII) Chelating agent：

Wherein Rc is the coupling moiety that the part that tissue is targetted is connected to by amide groups, preferably AGC0019.

17. the thorium complex of the tissue targeting of any one of claim 12 to 16, it includes the part that tissue is targetted, described group The part for knitting targeting is selected from monoclonal antibody or polyclonal antibody, antibody fragment (such as Fab, F (ab ')₂, Fab ' or scFv), Or the construct of this antibody-like and/or fragment.

18. the thorium complex of the tissue targeting of any one of claim 12 to 17, it includes the part that tissue is targetted, described group The part for knitting targeting includes at least one peptide chain, and the peptide chain has at least 90% sequence similar at least one following sequence Property：

Light chain：

DIQLTQSPSSLAVSAGENVTMSCKSSQSVLYSANHKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISRVQVEDLAIYYCHQYLSSWTFGGGTKLEIKR(SeqID1)

DIQLTQSPSSLASAAVEDRTMSCKSSQSVLYSANHKNYLAWYQQKPGQKAKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYLSSWTFGGGTKLEIKR(SeqID2)

Heavy chain：

QVQLQESGAELSKPGASVKMSCKASGYTFTSYWLHWIKQRPGQGLEWIGYINPRNDYTEYNQNFKDKATLTAD KSSSTAYMQLSSLTSEDSAVYYCARRDITTFYWGQGTTLTVSS(SeqID3)

QVQLQQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGYINPRNDYTEYNQNFKDKATITAD ESTNTAYMELSSLRSEDTAFYFCARRDITTFYWGQGTTVTVSS(SeqID4)

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGYINPRNDYTEYNQNFKDKATITAD ESTNTAYMELSSLRSEDTAFYFCARRDITTFYWGQGTTVTVSS(SeqID5)。

19. pharmaceutical preparation, it includes the thorium complex of the tissue targeting any one of at least one claim 12 to 18.

20. the pharmaceutical preparation of claim 19, it also includes citrate buffer agent.

21. the pharmaceutical preparation of claim 19 or claim 20, it is also included to aminobutyric acid (PABA) and optional EDTA And/or at least one polysorbate.

22. any one of thorium complex or claim 19 to 21 of the tissue targeting any one of claim 12 to 18 Described pharmaceutical preparation is preparing the purposes in being used to treat Hypertrophic or tumor disease medicine.

23. the purposes described in claim 22, wherein the disease is cancer, sarcoma, myeloma, leukaemia, lymthoma or mixed Mould assembly cancer, it includes NHL or B cell tumour, breast cancer, carcinoma of endometrium, stomach cancer, acute myeloid are white Blood disease, prostate cancer or the cancer of the brain, celiothelioma, oophoroma, lung cancer or cancer of pancreas.

24. the method that one kind treats the mankind or non-human animal (in particular for its mankind or non-human animal), it includes Give thorium complex or at least one claim that the tissue any one of at least one claim 12 to 18 is targetted Pharmaceutical preparation any one of 19 to 21.

25. the method for claim 24, methods described is used to treat Hypertrophic or tumor disease, such as cancer, sarcoma, marrow Knurl, leukaemia, lymthoma or mixed type cancer, it includes NHL or B cell tumour, breast cancer, endometrium Cancer, stomach cancer, acute myelogenous leukemia, prostate cancer or the cancer of the brain, celiothelioma, oophoroma, lung cancer or cancer of pancreas.

26. tissue targeting any one of thorium complex or claim 19 to 21 institute any one of claim 12 to 18 The pharmaceutical preparation stated, it is used to treat proliferative disease and/or tumor disease such as cancer, sarcoma, myeloma, leukaemia, lymph Knurl or mixed type cancer, including NHL or B cell tumour, breast cancer, carcinoma of endometrium, stomach cancer, Acute Meyloid Property leukaemia, prostate cancer or the cancer of the brain, celiothelioma, oophoroma, lung cancer or cancer of pancreas.

27. for the kit in the method for any one of claim 1 to 11, the kit is included：

I) four are included at N by C₁-C₃Alkyl-substituted Hydroxypyridinone (HOPO) partly with end be hydroxy-acid group coupling Partial octadentate chelating agent；

Ii) the peptide or protein matter of at least one tissue targeting comprising at least one amine moiety；

Iii) at least one am amide coupling agent；And

Iv) optional and preferred transmitting α thorium radionuclide, for example²²⁷Th。